Anal. Chem. 1985, 57, 1937-1941 (25) Brewer, C. F.; Riehm, J. P. Anal. Biochem. 1967, 78, 248-255. (26) Machida, M.; Sekine, T.; Kanaoka, Y. Chem. fharm. Bull. 1974, 22, 2642-2649. Sekine, T.; Kanaoka, Y. Chem. fharm. (27) Machlda, M.; Machida, M. I.; Bull. 1977, 25, 1678-1684. (28) Machida, M.; Machida, M. 1.; Kanaoka, Y. Chem. fharm. Bull. 1977, 25, 2739-2743. (29) Dnggan, D. E.; Bowman, R. L.; Brodie, B. 6.; Udenfriend, S. Arch. Blochlm. Blophys. 1957, 68, 1-14. (30) Imai, K.;Watanabe, Y. Anal. Chlm. Acta 1981, 130, 377-383.
1937
(31) Watanabe, Y.; Imai, K. J. Chromatogr. 1982, 239, 723-732. (32) Benson, J. R.; Hare, P. E. R o c . Natl. Acad. Sci. U . S . A . 1975, 72, 6 19-622. (33) Jones, B. N.; Gilligan, J. P. J. Chromatogr. 1983, 266, 471-482.
RECEIVED for review March 15,1985. Accepted April 24,1985. Presented in part at the 105th Annual Meeting of the Pharmaceutical Society of Japan, Kanazawa, April 3-5,1985.
Determination of Trimethylselenonium Ion in Urine by Ion-Exchange Chromatography and Molecular Neutron Activation Analysis Alan J. Blotcky and Gregory T. Hansen
Medical Research, V.A. Medical Center, Omaha, Nebraska 68105 Laura R. Opelanio-Buencamino and Edward P. Rack*
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
A method has been developed for the determlnatlon of the trlmethylselenonlum Ion (TMSe) In urine by anion exchangecation exchange chromatography, seiectlveiy capturing the TMSe on the cation exchange resin. TMSe recoverles are 91.9 f 7.6%. The TMSe fractlon is lrradlated wlth neutrons and radloassayed for 77mSeactivity. Varlous experlmental procedures Including acld hydrolysls, nltrlc acld digestlon, and HPLC were evaluated In order to optlmlze the determination.
The importance of trace selenium in living biological systems has been demonstrated in the literature (1-5). It has been shown that selenium is not only a vital micronutrient but is also a toxic agent at excessive levels. There is a narrow range of selenium intake that is consistent with health; outside of this narrow range, deficiency diseases and toxicity occur. Because of its biological importance various analytical procedures have been developed for analysis of microquantities of elemental selenium in serum and tissue (6,7). For urine these include atomic absorption spectrometry, solution absorption spectrometry, solution fluorescence spectrometry, volumetry, and neutron activation analyses (6-8). There is a paucity of the number of total selenium urine values in normal and disease states. Valentine et al. (8)found selenium urine concentrations with a mean value of 79.3 f 38.7 pg L-l for a sample size of 35 subjects, They suggest that selenium urine values correlate well with selenium water intake while serum values do not. The most promising technique for total selenium in urine appears to be neutron activation analysis (NAA) employing 75Se = 120 days), %e = 18 min) (9,lo),and 77mSe(Tl/z = 17.6 s) (11,12).The use of the 77mSe isotope is advantageous in that a large number of samples can be analyzed routinely if a nondestructive technique is employed. The metabolism of selenium in living organisms is undoubtably quite complex and the form of selenium which occurs within the living system depends on the form supplied (13).The possible metabolic interrelationship between organic
and inorganic forms of selenium has been described in the literature (14). The biological mechanisms mostly involve reduction and methylation. While little is known about the pathways by which the different forms of selenium are metabolized to trimethylselenonium (TMSe), they must involve a detoxification mechanism (15-19),TMSe is an important urinary metabolite at doses of selenite insufficient to trigger the respiratory excretion of dimethyl selenide (DMSe). Previous techniques to measure TMSe levels in urine involve the use of the radiotracer 75Se(9,15-19). Its use is limited because of the relatively long biological half-life of the selenium isotope and the associated issues of radiation exposure as well as constraints associated with handling of radioactivity. Several methods have been developed for the determination of total selenium and TMSe in urine. Nahapetian (19)employed a modification of the procedure described by Janghorbani (10) for the total determination of urine selenium. The selenium underwent several wet oxidation steps to Se(1V) with subsequent precipitation with ammonium pyrrolidinecarbodithioate (APDC) and its content measured either by fluorometric means or by neutron activation analysis. Palmer et al. (17) were the first to identify TMSe as a major excretory product in urine. These authors employed a cation-exchange-paper chromatography method for the separation and identification of 75Se-labeledTMSe. Nahapetian (18)employed either a modification of the Palmer method (16,17) for 75Se-labeled TMSe or anion exchange-cation exchange chromatography with subsequent wet oxidation conversion to Se(1V) and APDC precipitation. It was our intent to simplify the procedures for total selenium and TMSe determination in urine employing ITmSeactivation. A molecular neutron activation analysis procedure (MoNAA) such as that developed by this laboratory for the determination of iodoamino acids and hormonal iodine (20) and chlorinated pesticides (21) in a urine matrix has definite advantages over radiometric and fluorometric techniques. By use of 77mSeactivation the parts per billion (ppb) range can be readily attained. The major purpose of this study is to evaluate various separation procedures such as ion exchange
0003-2700/85/0357-1937$01.50/0 0 1985 American Chemical Society
1938
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
chromatography and liquid chromatography which can be coupled to neutron activation for the determination of TMSe in urine. EXPERIMENTAL SECTION Reagents, Solvents, and Resins. ACS reagent grade hydrochloric acid, nitric acid, glacial acetic acid, sodium sulfate, and ammonium nitrate from Fisher Scientific Co. (Fair Lawn, NJ) were used as received. Burdick and Jackson Laboratories, Inc. (Muskegon, MI), HPLC-grade acetonitrile, and methanol were used without further purification. Sodium selenite, seleno-DLcystine, seleno-DL-ethionine, seleno-DL-methionine, and 2mercaptoethanol were obtained from Sigma Chemical Co. (St. Louis, MO). Lithium nitrate, anhydrous 98.8%, was obtained from Alfa Products (Danvers, MA). Dimethyl selenide,obtained from Columbia Organic Chemical Co., Inc. (Camden, SC), was used to prepare trimethylselenonium according to the procedure described by Palmer et al. (16). Triply distilled water was used in all phases of the procedure. HPLC Instrumentation and Columns. An ISCO Model 1 4 0 liquid chromatograph (LC) with Model 314 metering pump and a Model UA-5 absorbance monitor (254 nm) with built-in recorder were used. The eluent which was to be radioassayed was collected employing an ISCO Model 328 fraction collector (collecting 1or 2 mL volumes in polystyrene sample cups). Three 6.35 X 250 mm Whatman (Clifton, NJ) columns were evaluated Partisil-10 ODS-2 (C,, reversed phase), Partisill0 (C, reversed phase), and Partisil5 (silica gel). The solvent reservoir, guard column, and the column itself were operated at ambient temperture. The mobile phase consisted of a 100 + 3 (v/v) water-acetonitrile mixture and was maintained at a flow rate of 1 mL/min and a pressure of 2100 psi. The mobile phase solvent was degassed prior to use and isocratic elution was employed. The ultraviolet detector was set at a wavelength of 254 nm. Resin Columns. The anion and cation exchange chromatography columns for evaluating the separation of the selenium and urine samples were prepared by loading a 0.7 X 10 cm borosilicate glass column with 6 g of Bio-Rad (Richmond, CA) AG-1-X8 resin in the acetate form (100-200 mesh) or AG-50W-X8 resin in the hydrogen form (200-400 mesh). The anion exchange chromatography columns for trapping the protein-bound selenium were prepared by loading a 0.7-4 cm Bio-Rad polystyrene column with 1.5 g of Bio-Rad AG-2-X8resin in the chloride form (200-400 mesh). Neutron Irradiation. Clear polystyrene (4 mL capacity) conical automated analyzer sample cups (Technicon Instrument Corp., Tarrytown NY) with polyethylene caps (Fisher Scientific, Pittsburgh, PA) were used for all HPLC eluent irradiations and 2-dram clear polyethylene vials with snap-on caps (Electro-Sonic Corp., Hawthorne, CA) were used for the ion exchange resin irradiations. Samples were irradiated for 20 s at a thermal neutron flux of 3.1 x loll n cm-2 s-l in the Omaha Veterans Administration Medical Center TRIGA reactor by means of a pneumatic transfer tube. Radioassay. All irradiated samples were allowed to decay for 20 s and counted for 20 s livetime utilizing a 60 cm3Ge(Li) detector (Princeton Gamma-Tech,Princeton, NJ) with an 11.3% efficiency. For comparison purposes two 5 mm X 3 in. NaI (Tl) detectors were used in parallel. These thin crystals directly reduce the sodium and chlorine radioactive contributions. Gamma spectra analyses were accomplished using a Nuclear Data (Schaumberg, IL) ND600 4096-channel analyzer. Preparation of TMSe. Trimethylselenonium chloride, Se(CH3)3+C1-was synthesized according to the method of Palmer et al. (17). Analysis of the synthesized compound by mass spectrometry indicated its purity as >98%. Total Selenium Determination in Urine. Several methods ( 8 , 2 2 )for the determination of total selenium in urine involve multiple steps including vigorous digestion and oxidation with HN03and HClO,, in order to ensure complete oxidation of urine to Se(1V)-Se(V1) with subsequent precipitation. In a previous study by this laboratory (11) total selenium was determined in biological specimens such as serum and tissue by an instrumental neutron activation procedure. In order to prevent possible losses of selenium in chemical operations, total selenium in urine was determined by instrumental neutron activation analysis (INAA)
0
77mseo32-
A
77m Se-Methionine 77m se-cystine
77mSe-Eth~oome
TMSe
I
10 15 ELUTION VOLUME (mU
20
Figure 1. Chromatogram of selenium compounds in aqueous solution. The HPLC mobile phase is water-acetontiie (100 + 3 (v/v)) employing a Partisil-10 ODs-2 column.
employing 3-mL quantities of unprocessed urine. Neutron irradiated and radioassay procedures were as previously described. Determination of TMSe. The procedure for the determination of TMSe in a urine matrix involves passing a volume (2-10 mL) of unprocessed urine through anion- and cation-exchange chromatographic column in tandem and then eluting the TMSe with 0.5 M LiN03. The purpose of the anion exchange column was to trap the peptide linked selenium allowing the TMSe, as a cation, to be selectively trapped in the cation exchange resin. The radioactivable elements, sodium and chlorine, are eluted through the resin. The resin, containing the TMSe, is allowed to dry overnight, transferred to a polyethylene vial, and analyzed for 77mSeby neutron activation analysis. RESULTS AND DISCUSSION Liquid Chromatograph Separations i n Aqueous Solutions. Three LC columns, Partisil-10 ODs-2, Partisil 10 (C, reversed phase), and Partisil5 (silica gel), were evaluated for the separation of TMSe from seleno amino acids and radioactive sodium and chloride ions. The Partisil-10 ODs-2 column offered the best separation of TMSe from the seleno amino acids. This can be seen in Figure 1. Various elution solutions were employed to give the best separation of TMSe from other species. Because TMSe is an ion it is eluted as a rather sharp peak between 1.5 and 2.5 mL of elution solution regardless of the column employed. Liquid chromatography can be employed in the separation and identification of unknown metabolites in urine. Prior to LC separation, the raw urine must be processed by acid digestion, hydrolysis, or solvent extraction, as discussed by us previously (20, 21). Cation Exchange Separation in Aqueous Solutions. employing a Bio-Rad AG-50W-X8 cation exchange column, various elution solutions were evaluated for the separation of TMSe from a urine matrix. The 0.5 M LiN03 solution demonstrated excellent separation of radioactivable sodium and chloride ions, selenite, ion, and biologically important seleno amino acids. The interesting separation characteristic was that all the above species investigated eluted before TMSe. The separation characteristics are presented in Table I. The recovery yields were determined by collecting the elution solution in separate 2-mL vials, irradiating them with neutrons, and radioassaying for lImSeactivity. It was found that for TMSe and all the seleno amino acids studied, the recoveries were relatively high and constant between 93 and 100%. Because of the distinct separation between TMSe and
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
Table I. Separation Characteristics of Seleno Organic Compounds, Sodium Selenite, TMSe, and Sodium Chloride in an Aqueous Matrix Employing Cation Exchange Chromatography" compound/ion
peakb width, mL
selenomethionine selenoethionine selenocystine sodium selenite Na+ ion C1- ion trimethylselenonium ion
44
45 43
Table 111. Separation Characteristics of Seleno Organic Compounds, Sodium Selenite, and TrimethylselenoniumIon in a Hydrolyzed"Urine Matrix Employing Cation Exchange Chromatographyb
yield: %
compound/ion
peakc
100
selenomethionine selenocystine sodium selenite Na+ ion C1- ion trimethylselenonium ion
6 6
5
100 93
4
3
94
39 3 198
5
100 100 100
2 62
"Bio-Rad AG-50W-X8 column eluted with 0.5 M LiN03. bNumber of vial (each vial contains 1 mL of eluent). cAverage (A) standard deviation for six replicate determinations. Table 11. Effect of Nitric Acid on Recovery of Total Selenium in Urine with Added TMSe %
sarnpleavb 1. dry on sand bath bring up in 2 mL of HN03 (concn) 2. dry on sand bath redissolve in 2 mL of 1 M HNO,
redry to remove Cl bring up in 2 mL of HN03 (concn) 3. dry on sand bath redissolve in 2 mL of 1 M HN03 redry to remove C1 bring up in 2 mL of 1 M HN03 4. dry on sand bath with 10 mL HN03 (conc) added bring up in 2 mL of HN03 (concn)
recoverf 99d 99d
99d
99'
"All samples contain 10 mL of urine plus 1 mL of TMSe. bAll samples heated on sand bath at 60-65 O C . n = 6. dSamples analyzed for total 77mSeby INAA procedure. 'TMSe separated employing a cation exchange column. the other compounds it was decided to collect the TMSe on the resin rather than to elute it as previously described by others (1419)). It would take approximately 200 mL to elute completely all of the TMSe from the resin. This would then require a concentration step with potential loss of TMSe. Digestion and Hydrolysis of Urine. Although TMSe is not protein bound as are seleno amino acids, it would also be important to discuss the effects of digestion and hydrolysis on TMSe and on seleno amino acids especially in developing the liquid chromatographic procedures. Janghorbani et al. (10) and Nahapetian et al. (19) describe the quantitative conversion of TMSe to the SenIV) oxidation state with various oxidant mixtures for the purpose of separating selenium in urine. They found that TMS is not oxidized to any significant extent by HN03 digestion. The effect of nitric acid digestion on the recovery of total selenium in urine with added TMSe is presented in Table 11. As a confirmation of previous results (10,19),the percent recovery of total selenium and TMSe is quantitative at 99%. However, seleno amino acids such as selenomethionine undergo decomposition with HN03 digestion. For example, selemethionine recovery after HN03 digestion is about 55%. The effect of hydrolysis on TMSe and several seleno amino acids in a urine matrix was evaluated employing a cation exchange chromatography column. These results are shown in Table 111. While there is an excellent separation of TMSe from other species, the recovery yields are quite variable for TMSe ranging from 28 to 84%. It is interesting to note that the recovery yields of selenomethionine and selenocystine are quantitative and greater than 90%. Determination of Total Selenium in Urine. On the basis of our experience with 77mSeactivation of various biological
1930
width, mL
% yieldd
84
24
68 32 36 5 110
48
97 A 3 90 f 4 72 f 3
28
20 10 122
28-84
"Five milliliters of urine, 3 mL of 6 M HC1, 2 mL of glacial acetic acid, and 0.5 mL of 1 M mercaptoethanol were sealed in a glass ampule and heated to 100 O C for 5 h. b A Bio-Rad AG-BOWX8 column eluted with 0.5 M LiN03. cNumber of vial (each vial contains 1 mL of eluent). d n = 6. matrices, it is our contention that an INAA procedure could be best employed for the quantitative determination of total selenium in urine. By use of a nondestructive method, loss of selenium due to digestion, hydrolysis, volatilization, lyophilization, or other handling procedures is minimized. As will be discussed later, the results are quite quantitative. Optimum Procedure. A suitable technique for the determination of TMSe could involve separation of TMSe from the urine matrix by liquid chromatography or ion exchange chromatography. Because TMSe is not decomposed by nitric acid digestion, urine samples could be digested with concentrated nitric acid to dryness, brought up in elution solution, and separated by LC and the vials containing TMSe irradiated with neutrons and radioassayed for 77mSe. The optimum procedure obtained for TMSe in urine matrix involves passing 6.5 mL of unprocessed uring through an anion exchange chromatographic column and a cation exchange chromatographic column in tandem and eluting with 97 mL of 0.5 M LiN03 The purpose of the anion column is to trap the peptide linked seleno amino acids, allowing the TMSe, as a cation, to pass through to the cation exchange column while it is separated from the radioactivable Na+, C1-, and Se032-and non-protein-bound seleno amino acids. In order to test the efficiency of the combination columns for separating out peptide linked complexes onto the anion column, allowing TMSe to be captured on the cation exchange chromatographic column, 6.5 mL of raw urine, spiked with 10 &i of 1311-labeled serum albumin (1 mg) and 0.1 ppm TMSe were passed through the two columns. No I3lIactivity passed through the cation exchange resin after the elution of 150 mL 0.5 M LiNO, solution. The recovery of TMSe on the resin was quantitative with an average yield greater than 90%. A typical elution pattern of seleno amino acids and TMSe in a urine sample with these additives, employing the two-column separation, is shown in Figure 2. As can be seen the elution of TMSe through the cation exchange column requires greater than 125 mL of elution solution. Rather than separate out the TMSe from the column and employing concentration steps as reported previously (191,TMSe was collected on the AG-50WX8 resin after elution with 97 mL of 0.5 M LiN0, solution. Presented in Table IV are the recoveries of TMSe in urine at varying TMSe concentrations. As can be seen in Table IV, the TMSe recoveries are quantitative and independent of TMSe concentration. Unlike the determination of aluminum in urine (23)where trace aluminum was recovered in the cation exchange resin (with the A1 in the parts-per-billion range), there are no detectable impurities of selenium in the resin. Depicted in Figure 3 is a y-ray spectrum, following neutron irradiation, of the resin after elution of the urine sample spiked with 1ppm TMSe. Elements other than selenium are naturally present in the resin.
1040
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
1
2oF
I
-i 80
1
I
1
I
I
500
1000
1500
1800
CiAMdA FAY PERGY, KeV
I
0
20 40
80
120
160 ELUTION VOLUME (mL)
200 220
Figure 2. Chromatogram of human urine spiked with selenium compounds employing neutron activation detection of '"'"Se after an elution with 0.5 M LiNO, solution through the tandem ion exchange chromatography columns.
Flgure 3. y-Ray spectra following neutron irradiation of urine passed
through an AG-50W-X8 resin. Table V. Total Selenium and TMSe in Urine sample
normal diet
Table IV. Recovery (%) of TMSe in Urine at Varying TMSe Concentrations Employing Anion Exchange and Cation Exchange Chromatography Columns in Tandem TMSe concn, fig mL-'
recovery,"* %
% RSD
55.0 27.5 13.8 6.9 3.4 1.8 0.9 0.4 0.2 0.1
87.5 f 4.4 91.2 f 0.7 91.9 f 5.0 99.2 f 2.5 101.9 f 6.3 99.7 f 1.7 88.9 f 4.2 96.7 f 4.2 83.4 f 9.6 78.6 f 9.9
5.0 0.8 5.4 2.5 6.2 1.7 4.7 4.3 11.5 12.6
mean
91.9 f 7.6
8.3
a Average f standard deviation for six replicate determinations. *Percent recovery measured with respect to standard. "MSe captured in AG-50W-X8 resin after elution with 97 mL of 0.5 M LiNO,.
Determination of Total Selenium and TMSe in Normal Urine. By use of the optimum procedure total selenium and TMSe in urine were determined in samples collected from two subjects: one having a normal diet and the other having a diet supplemented with selenium-containing vitamins (25 pg of Se/day). Presented in Table V are the assays. For levels of total selenium in the ranges reported in Table V, it would appear that a relative standard deviation no better than 35% can be expected employing the Omaha V.A. Medical Center nuclear reactor at its current neutron flux. However, by use of reactors with greater neutron fluxes, the relative standard deviation may be reduced accordingly. Consistent with previous TMSe determinations of urine from normal subjects, TMSe is not a major product, representing only about 30% of the selenium content. While he relative standard deviation appears rather high (25-35%), it must be realized that the levels of selenium and TMSe in normal urine are quite low. These are the first reported results of TMSe levels in normal subjects. It is our contention that trace levels of selenium and TMSe in urine can be routinely analyzed by the procedure described in this paper. It appears that by employing the reactor at the current reactor flux, a lower limit of detection
normal diet plus 25 pg of Se/day vitamin supplement
total selenium: pg mL-'
[TMSe]: fig mL-'
0.10 h 0.04 0.13 h 0.03
0.03 f 0.01 0.04 f 0.01
'Six separate determinations were made on the same urine collection. of 10 ppb for total selenium and TMSe can be obtained. Registry No. TMSe, 25930-79-4; Se, 7782-49-2; 77Se,1468172-2; Na, 7440-23-5; C1, 16887-00-6; nitric acid, 7691-37-2; selenomethionine, 2578-28-1; selenoethionine, 257827-0; selenocystine, 2897-21-4; sodium selenite, 10102-18-8.
LITERATURE CITED (1) McConnell, K. P.; Broghamer, W. L., Jr.; Blotcky, A. J.; Hurt, 0. J. J. Nutr. 1975, 705,206. (2) Broghamer, W. L., Jr.; McConnell, K. P.; Blotcky, A. J. Cancer 1978, 4 7 , 1462. (3) Broghamer, W. L., Jr.; McConnell, K. P.; Biotcky, A. J. Cancer 1976, 37, 1384. (4) Shamberger, R. J.; Rukovena, E.; Longfeld, S. A.; Tylko, S.;Deodhar, C. E.; Willis, C. E. Natl. Cancer Inst. 1973, 50,863. (5) Willett, W. C.; Polk, B. F.; Morrls, J. E.;et al. Lancet 1983, July 16, 130. (6) Iyengar, G. V.; Koiimer, W. E.; Bowen, H. J. M. "The Elemental Composition of Human Tissues and Body Fluids"; Verlag Chemie, New York, 1976. (7) Versieck, J.; Cornelis, R. Anal. Chim. Acta 1980, 776,217. (8) Valentine, J. L.; Kang, H. K.;Spiney, G. H. Envlron. Res. 1978, 77, 347. (9) Janghorbani, M.; Ting, 8. T. G.; Young, V. R. Am. J . Clin. NuQ. 1981, 34,2816. 10) Janahorbani, M; Tina, B. T. G.: NahaDetian, A,; Young, V. R. Anal. ' Ch&. 1982, 54, 1768. 11) Blotcky, A. J.; Arsenault, R. E.; Rack, E. P. Anal. Chem. 1973, 45, 1056.12) McKown, D. M.; Morris, J. S . J. Radioana!. Chem. 1970, 43,411. 13) Gunther, H. E. Biochemistry of Selenlum. Selenium" Zlngaro. R. A., Cooper, W. C., Eds.; Van Nostrand-Reinhold: New York, 1974; pp 546-608. 14) Sunde, R. A,; Hoekstra, W. G. Blochem. Siophys. Res. Commun. 1980, 93, 1181. 15) Foster, S. J.; Ganther, H. E. Anal. Biochem. 1984, 137,205. 16) Palmer, L. S.;Fischer, D. D.; Halverson, A. W.; Olson, 0. E. Biochlm. Slophys. Acta 1969, 777, 336. (17) Palmer, L. S.;Gunsalus, R. P.; Halverson, A. W.; Olson, 0. E. Biochim. Siophys. Acta 1970, 208. 260. (18) Nahapetian, A. T.; Janghorbani, M.; Young, V. R. J. Nutr. 1983, 713, '
401.
(19) Nahapetian, A. T.; Young, V. R.; JanghorbanC M. Anal. Blochem. 1984. 140, 56. (20) Firouzbakht, M. L.;Garmestani, S. K.; Rack, E. P.; Blotcky, A. J. Anal. Chem. 1981, 53, 1746. (21) Opelanio, L. R.; Rack, E. P.; Blotcky, A. J.; Crow, F. W. Anal. Chem. 1903, 55,677.
Anal. Chem. 1905, 57, 1941-1943 (22) Wetkinson, J. S. Anal. Chem. 1966, 38, 92. (23) Blotcky, A. J.; Hobson, D.; Leffler, J. A.; Rack, E. p. Anal. them. 1976, 48, 1084.
RECEIVED for review March 8,1985. Accepted April 22,1985.
1941
This research was supported by the U S . Department of Energy, Division of Chemical Sciences, Fundamental Interaction Branch, under Contact DE-FG02-MER13231 and a University of Nebraska Research Council NIH Biomedical Research Support Grant No. RR-07055.
Cobalt Preconcentration on a Nitroso-R Salt Functional Resin and Elution with Titanium( I I I) Renato Stella,* M. T. Ganzerli Valentini,* and Luigino MaggiO
Dipartimento di Chimica GeneraleO e Centro di Radiochimica ed Analisi per Attivazione del CNR, Universitci di Pavia, Viale Taramelli 12,27100 Pavia, Italy
The anion exchange resin Dowex 1x8, converted to the nitroso-R salt form, was used for adsorbing cobalt from large freshwater samples. Strongly acid titanium( 111) chloride M solution was found very effective at 60 O C as a new eluant and yielded complete recovery with a preconcentratlonfactor of 100. Subsequent atomic absorption spectrometry determinatlon of cobalt In the eluate was possible with no interference from titanium, reduced organics, and Iron, copper, and nickel which partially might be fixed onto the resin. The suggested procedure allows a reproducibility of 5-10 % for samples with cobait concentrations in the range of 0.01-1 Mg L-1.
Trace elements are usually present in freshwater a t microgram per liter levels. Their direct analysis is highly desirable as it involves minimum sample handling and pretreatment but it is limited to a few elements. Especially, cobalt, whose concentration in natural waters is generally below 1 pg L-l, unfortunately requires preconcentration; its measurement in freshwater is important to cope with the increasing demand for control of nuclear plant discharges. Several batch techniques have been used for cobalt preconcentration including coprecipitation (1,2)and cocrystallization (3),which yield high preconcentration factors but are tedious and time-consuming. Flow methods are simpler and convenient: usually they involve the use of ion exchangers (4),also in the form of membranes (5),or chelating exchangers loaded onto different inert supports (6-10). In some cases the adsorption of complexes on ion exchange resins was reported (11). A highly specific reagent for cobalt complexation, sodium l-nitroso-2-naphthol-3,6-disulfonate, also called nitroso-R salt (NRS), is widely used (2,10, 11). Moreover it has the advantage of bearing ion exchangeable sulfonato groups. In this work NRS was used to develop a simple method for preconcentrating cobalt from natural waters, based on its adsorption on an ion exchange resin previously converted to a functional NRS form. The development of these functional resins is due to Tanaka et al. (12)and to Lee et al. (13). Cobalt may be recovered either by removing the chelate from the resin bed or by displacing the metal ion from chelating groups; we found that, whichever the mechanism involved, the elution is the most critical step in the whole 0003-2700/85/0357-1941$01.50/0
procedure. Preliminary experiments showed in fact that, unlike that claimed by other authors (lo),any strong mineral acid was required in a quite large volume for a complete recovery and no substantial improvement was obtained by using any of the common oxidizing or complexing agents (HzOz, Clz, NH3, CNS-, EDTA). Reduction of nitroso group -NO with TiC13 in HC1 to the amino group -NH2 was much more effective and ensured a concentration factor of 100. Subsequent atomic absorption spectrometry measurements of cobalt was found unaffected by the presence of titanium and leached organic, mostly l-amino-2-naphthol-3,6-disulfonic acid. The preconcentration procedure was particularly devised for freshwater samples which usually bear cobalt in the concentration range 0.01-1 pg L-l. EXPERIMENTAL SECTION Reagents. Titanium trichloride TiCl,, from Merk Co., did not require purification and was added to Suprapure HCl (C. Erba) to reach a 4 M final HCl concentration. Sodium hydrogen carbonate, nitroso-R salt, and other chemicals were reagent grade. Terdistilled water was used in each experiment. Radiotracer “To (t,,,,= 5.3 years) was prepared by irradiating 10 mg of cobalt(I1) nitrate in a TRIGA MARK I1 reactor at the University of Pavia at a thermal neutron flux of 8 X 10I2 n cm-2 s-l for 60 h; the irradiated Co salt was dissolved and repeatedly diluted with distilled water and then with filtered river water to reach a Co concentration twice the natural Co level, while still giving a count rate of 10000 cpm mL-’. Anion exchange resin Dowex 1X8,100-200 mesh, was washed with 1M HCl and 1M NaOH and terdistilled water prior to use. Apparatus. A peristaltic pump (Millipore Co.) was used to feed the column, the latter being equipped with a water jacket to allow operation in thermostated conditions. Column dimensions were 0.8 cm i.d., 9 cm height. Radiotracer (60Co)was y counted by a NaI (Tl) well type crystal coupled to a multichannel analyzer. A Perkin-Elmer 2380 atomic absorption apparatus equipped with HGA 74 graphite furnace was used for Co determination. Resin Preparation. The functional chelating resin was prepared by supending 30 g of purified Dowex 1x8 resin, 1c0-200 mesh, in 100 mL of a solution containing 0.3 g of NRS. After the mixture was shaken for 1h, the resin was filtered on a glass filter under gentle suction and dried in a vacuum desiccator; NRS loading resulted in 20 rmol per g of resin. Procedure. Large Po River water samples were submitted, soon after sampling, to a multistep filtration using Millipore membrane packets of decreasing porosity, 8-0.45 rm, to minimize filter clogging. Filtered water aliquots (5 L) were treated with Na2C03and NaHCO, to adjust pH to 8, and fluxed through a water jacketed 0 1985 American Chemical Society
