27
Anal. Chem. 1884, 56, 27-29
The calibration curves for the determination of prednisolone and hydrocortisone were made on the basis of the peak heights of the corresponding steroids. Recovery of these steroids from the sera was approximately quantitative and reproducible as shown by a comparison of the injection of the standard sample solutions and sera containing known amounts of steroids (1-100 ng/mL serum). We applied the technique presented in this paper to the nano level analysis of serum corticosteroids such as hydrocortisone suppressed by prednisolone therapy for colagen disease and especially SLE patients. The results in Figure 2 show that our method is quite adequate for clinical assay before the use of prednisolone therapy or medication design determined by pharmacokinetics.
Nippon Medical College, for his suggestions and comments. Registry No. Cortisol, 50-23-7;prednisolone, 50-24-8; prednisone, 53-03-2; corticosterone, 50-22-6; cortisone, 53-06-5.
ACKNOWLEDGMENT The authors express their appreciation of M. Shibuya,
RECEIVED for review October 13,1982. Resubmitted June 28,
LITERATURE CITED (1) Hara, S.; Oka, K.; Dobashi, Y.; Ohkuma, T. Yakugaku Zasshi 1982, 102, 107-109. 12) . . Oka, K.; Dobashi, Y.; Ohkuma, T.; Hara, S . J. Chromatogr. 1981, 217, 387-398. (3) Hara, S.; Ijitsu, T.; Oka, K. Yakugaku Zasshi f982, 102, 895-897. (4) Hara, S.;Ijltsu, T.; Dobashi, Y.; Oka, K. Yakugaku Zasshi 1982, 102, i .oss-iodci - - - .- , -. (5) Hara, S. US. Patent 4 289 620, 1981. (6) Rose, J. Q.; Jusko, W. J. J . Chromatogr. 1979, 162, 273-280. (7) Engelhardt, H.;Mueller, H. J. Chromatogr. 1981, 218, 395-407. (8) Hara, S.; Dobashl, Y.; Oka, K. J. Chromatogr. 1982, 239, 677-685.
1983. Accepted September 9,1983.
Preconcentration with Dithiocarbamate Extraction for Determination of Molybdenum in Seawater by Neutron Activation Analysis W. M. Mok and C. M. Wai* Department of Chemistry, University of Idaho, Moscow, Idaho 83843
Molybdenum in seawater can be quantltatively extracted with pyrrolldlnedlthlocarbamate and dlethyldlthiocarbamate at pH 1.4 into chloroform, for neutron activatlon analysis. Uranium in seawater cannot be extracted at this pH, and hence ellmMo reaction. Inhates the interference from the 23sU(n,f)gg terferences from matrlx specles in seawater, such as sodlum and bromlne, are also removed durlng the extraction. The proposed method, with good accuracy and sensitivity, Is sultable for the determlnatlon of molybdenum in natural waters.
The determination of certain trace elements has become important in studies of marine environments. Among these, molybdenum, as a biologically active element for growth and as a micronutrient in the aquatic environment, is often measured. Because of the low levels of molybdenum involved, neutron activation analysis (NAA) appears to be a suitable technique. Molybdenum can be identified in NAA through %Mo (tlIz= 66 h) or ita daughter 99”Tc (tl12= 6.05 h). Direct application of NAA for trace element determination in complex systems is generally difficult because of serious matrix interferences. Chemical separation methods have been used either before or after neutron irradiation to eliminate such interferences. Besides matrix interferences, another complication in the molybdenum determination by NAA is the contribution of 99Mofrom the 23SU(n,f)94Moreaction, which is significant in systems with high uranium contents. Separation before neutron irradiation has the advantage of avoiding this complication if the method is selective for molybdenum. Kulathilake and Chatt reported a preconcentration method for the NAA determination of molybdenum in seawater by cocrystallization with P-naphthoin oxime (1). This method
is selective for molybdenum but requires a couple of days to complete the separation. Molybdenum has also been determined in seawater after preconcentration on activated charcoal in the presence of ammonium pyrrolidinedithiocarbamate (APDC), followed by neutron activation of the charcoal sample (2). Since activated charcoal is an efficient adsorber, a significant fraction of the uranium in seawater was also found with the molybdenum in the charcoal. Consequently, a correction for the uranium contribution to 99Momust be considered in this charcoal adsorption method. This paper describes a simple solvent extraction method using dithiocarbamates for preconcentration of molybdenum in seawater. The solvent extraction method not only is fast and efficient but also eliminates the interferences caused by uranium and other matrix species in the determination of molybdenum by NAA. EXPERIMENTAL SECTION Ammonium pyrrolidinedithiocarbamate (APDC) and sodium diethyldithiocarbamate (NaDDC) were obtained from the Fisher Scientific Co. Chloroform used in the extraction was Baker Analyzed Reagent. Nitric acid used in the experiments was of Ultrex grade from Baker Chemical Co. Deionized water was obtained by treatment of distilled water through an ion exchange column (Barnstead Ultrapure Water Purification Cartridge) and a 0.2-pm filter assembly (Pall Corporation Ultipor DFA). A stock solution containing 1000 pg/mL molybdenum was prepared by dissolving 1.8401 g of dry ammonium molybdate (analytical reagent grade) in deionized water and diluted to a liter in accordance with the EPA’s instructions (3). Standard working solutions were prepared by appropriate dilution of the stock solution. Surface seawaters were collected from the southwest corner of Discovery Park in western Seattle. The salinity of the seawater was 27%0.The seawater was filtered through a 0.45-pm membrane filter, acidified to pH 2, and stored in precleaned polyethylene bottles. All containers used in this study were
0003-2700/84/0356-0027$01.50/00 1983 American Chemical Society
28
ANALYTICAL CHEMISTRY, VOL. 56, NO. 1, JANUARY 1984
washed with 10% nitric acid, rinsed with deionized water, and stored in a class 100 clean hood equipped with a vertical laminar flow HEPA filter. The extraction solution was prepared by dissolving 1g of APDC and 0.5 g of NaDDC in 100 mL of deionized water. In all cases, the extraction solution was prepared fresh daily, filtered to remove the insoluble material, and shaken for 30 s with chloroform to remove bromine and other impurities. In the determination of molybdenum in seawater, a 100-mL sample of the filtered seawater was poured into an Erlenmeyer flask, equipped with a ground stopper. The sample was adjusted to pH 1.4 0.1 before 2 mL of the extraction solution and 5 mL of chloroform were added. The mixture was shaken for 15 min, and then was allowed to sit for 10 min for phase separation. After the waiting period was up, 3 mL of the organic phase was collected and placed in a polyethylene vial. The solution was evaporated to dryness before heat-sealing the vial for neutron irradiation. The preconcentration factor attained with this procedure is 60. Standards were made of 1-mL solutions containing proper concentrations of molybdenum, sealed in the same kinds of vials as the samples. One hundred milliliters of deionized water that went through the same extraction procedure was used as a blank for seawater analysis. All samples and standards were normally irradiated for 2 h in a 1-MW TRIGA reactor at a steady neutron flux of 6 X 10l2n cm-2 s-l. After irradiation, the samples were allowed to decay for about 48 h, to allow 99Moand 99mTcto reach equilibrium before counting. The following sample transfer procedure was used to avoid the interferences of %Naand other radioactivities produced in the plastic material of the irradiated vials. To each vial, 1 mL of a 3 N HN03 solution was injected by means of a 5-mL disposable syringe. After shaking for about 1min, the acid solution, containing any molybdenum present, was transferred, using the same syringe, into a new 2/5-dram vial for y counting. Less than 1% of the molybdenum was found to remain in the irradiated vial after sample transfer. Standards were also transferred into new 2/5-dramvials with disposable syringes. Each sample was counted 2 X lo3 s with a large volume coaxial ORTEC Ge(Li) detector with a resolution of about 2.3 keV for the 1332-keV y-ray of %o, a peak-to-Compton ratio of 351, and an efficiency of 11%. The detector output was fed into a Nuclear Data 4096-channel was used in pulse-height analyzer. The 140-keV y-ray of 99mT~ this work to determine the molybdenum concentration. Data analysis was carried out by using the SPAN program with an IBM 370 computer. Experiments were also carried out to determine the extracted molybdenum by graphite furance atomic absorption spectrometry (GFAAS). In this case, the dried sample was first dissolved in 1 mL of concentrated nitric acid, and then diluted to about 2% of the acid. A Perkin-Elmer HGA 2100 graphite furnace and a Model 603 AA spectrometer were used for this study. PerkinElmer pyrolytically coated graphite tubes were used, and a deuterium-arc background correction was applied. The operation conditions for molybdenum determination followed that prescribed by the EPA (3). A 25-pL Eppendorf micropipet was employed for injecting samples into the furnace. All samples and standards were injected in triplicate, or more if triplicates disagreed significantly. Standards spanning the sample absorbance range were run between each series of samples as a regular check.
RESULTS AND DISCUSSION Conditions for the Extraction of Molybdenum from Seawater. Extraction of metal ions by dithiocarbamate depends on several factors, such as type of solvent, amount of dithiocarbamate, pH of a solution, and shaking time. The advantages of using a mixture of APDC + NaDDC, and chloroform as solvent, to extract trace metals from seawater have been described in the literature (4). APDC is more stable than NaDDC in acid solution, but the mixture provides a better working pH range and appears to have a stabilizing effect on metal complexes. In this study, a mixture of 2:l APDC to NaDDC, at a total concentration of 0.03% or greater, was used for molybdenum extraction. To test the effect of pH on the extraction, a synthetic seawater whose composition was described in a previous paper was spiked with 48 pg/L
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pH dependence of the extraction of molybdenum from seawater by APDC NaDDC into chloroform. Figure 1.
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pH dependence of the extraction of urantum from seawater by APDC (open circles) and by NaDDC (closed circles). Figure 2.
of molybdenum ( 4 ) . Ten-milliliter aliquots of this spiked seawater were taken, with the pH adjusted to different values. The spiked molybdenum in each sample was extracted with the dithiocarbamate solution into 2 mL of chloroform for NAA. A blank of unspiked synthetic seawater was run in the same manner as the samples in each irradiation. In all cases, the blanks showed less than 1% of molybdenum activities relative to the samples. After correction for the background activities, the percentages of molybdenum recovery for the spiked seawater samples are shown in Figure 1. Near total recovery of molybdenum was observed from pH 0.7 to 4.0. At pH 4.6, recovery of molybdenum was found to decrease to about 7 5 % . Further increase in pH resulted in very poor recovery of molybdenum. Similar experiments with the natural seawater spiked with 48 pg/L of molybdenum showed almost identical pH dependence. Extraction of molybdenum at very low pH is not practical because of the accelerated decomposition of dithiocarbamates in acid solution (5). On the other hand, extraction a t pH >2 is also not satisfactory because uranium in seawater can be extracted above this pH (Figure 2). Therefore, a pH range of 1.3 to 1.5 was chosen as the standard condition for this method. Under our experimental conditions, extraction of molybdenum for seawater is kinetically a very fast process. For 10 mL of the spiked seawater with 1mL of the APDC + NaDDC solution and 2 mL of chloroform, extraction of molybdenum is virtually complete in 1min of vigorous shaking. With a large volume of seawater, e.g., 100 mL of sample with 2 mL of the extraction solution and 5 mL of chlorofQrm, several minutes of shaking is sufficient to complete the extraction. Extraction of Uranyl Dithiocarbamate Complexes. The interfering nuclear reactions in the molybdenum determination by NAA are 120Ru(n,a)ggMo and 235U(n,f)99Mo.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 1, JANUARY 1984
However, the amount of ruthenium in natural waters is so low that its contribution to 99Mois negligible. The 235U(n,f)wMo reaction is important in samples in which the ratio of Mo:U is close to one ( 2 , 6 ) .The average uranium concentration in seawater is about 3.2 ppb, comparable to that of molybdenum in seawater (7). Therefore, the contribution from fission of 235Uneeded to be investigated under the proposed extraction conditions for molybdenum. The various effects of pH on the extraction of uranium in seawater by APDC and by NaDDC are shown in Figure 2. The experiments were carried out with a synthetic seawater spiked with 50 pg/L of uranium, extracted with either APDC or NaDDC into chloroform, following a procedure similar to that described for molybdenum. The extracted uranium was determined by NAA, utilizing the 228-keV y-ray from the decay of 23gNp(daughter of 239U,tllz = 2.36 days). As shown in Figure 2, extraction of uranium with either APDC or NaDDC depends strongly on pH. At pH 4 and reaches a narrow maximum at a pH of around 5.5-6.0. The uranyl-PDC complex is apparently stable in slightly acidic solutions, whereas the corresponding DDC complex is stable in near neutral solutions. In either case, extraction of uranium is insignificant at a pH of around 1.3-1.5. This is also true when a mixture of APDC and NaDDC, in a ratio of 2:1, was used in the extraction. The recommended pH for molybdenum extraction should be totally free of uranium interference. Determination of Molybdenum by NAA. One advantage of using dithiocarbamate for preconcentration of trace metals in seawater is that matrix species such as the alkali metals, the alkaline earth metals, the halogens, and phosphorus can be simultaneously eliminated during extraction. In a previous study of preconcentrating gold and mercury from seawater for NAA, it has been shown that sodium and bromine in the system can be essentially all removed with dithiocarbamate extraction (8). The large amount of 24Naand szBr produced in seawater upon neutron irradiation would otherwise mask the low-energy peaks from radioisotopes such as %Moor 99”Tc. Removing these matrix species, as well as uranium, from the system enables accurate determination of molybdenum in seawater without spectral interference. The procedures for molybdenum extraction and NAA described in this paper have been applied to the analysis of NBS Standard River Water (SRM 1643a), which is certified to contain 95 f 6 ng/g of molybdenum. Based on four replicate analyses, the average molybdenum content determined by this extraction method and NAA was 102 f 4 pg/L, which agrees well with the certified value. The precision of this method for seawater analysis was tested by individually preparing seven samples, all at pH 1.4 f 0.1 and each containing 48 pg/L of molybdenum. The average recovery was 100.2% , and the average coefficient of variation for the series of replicates was f3.5%. With 100 mL of seawater treated by the described procedure, the detection limit, based on three standard deviations of the background under the 140-keV peak, in a counting period of 2 x lo3 s and the specified irradiation conditions, was estimated to be 0.35 pg/L. This is sensitive enough for molybdenum determinations in seawaters. The detection limit can be further improved by increasing the
29
preconcentration factor, the irradiation time, and the counting time. The molybdenum content in the seawater collected from the Seattle coast was determined by NAA, after preconcentration with this extraction method, to be 8.8 f 0.3 pg/L. The ratio of molybdenum concentration (in pg/L) to salinity (in %DO) for this sample is 0.33, which is consistent with the average ratio of 0.32-0.34 found in open ocean waters (9, 10). Determination of Molybdenum by GFAAS. Four replicate analyses of NBS Standard River Water (SRM 1643a) were also carried out using this extraction method and GFAAS. The average molybdenum content was found to be 97 f 6 pg/L, in good agreement with the value obtained by NAA. The average molybdenum concentration in the seawater from Seattle was determined by GFAAS to be 9.0 f 0.4 pg/L, which again is in good agreement with the value of 8.8 f 0.3 pg/L determined by NAA. In this GFAAS study, samples and standards were all kept in 2% nitric acid solution. Our experiments indicate that the effect of acid concentration on the signal of molybdenum absorbance is small (
