Standards were produced by precipitating known amounts of Au in the 0- to 12-pg range as the sulfide. Measurement of the Au La line was made as described above. The calibration curve produced was linear, passes through the origin, extends to -28,000 counts for 10 pg Au and is defined by the equation, y = 2838x - 33. All eight calibration points lie close to the line. The linearity suggests that the precipitation of Au is complete. T o ascertain whether gold is lost during the digestionprecipitation-filtration processes, an experiment was carried out to test the recovery of gold. Various amounts of gold were added to serum samples and the samples were then put through the complete procedure. The results of these recovery experiments are presented in Table VI. In general, the recovery was reasonably good with the majority of the results clustering within f 6 % of the correct value. The results show that X-ray fluorescence analysis can be used as a rapid and accurate method for bromine and iodine analysis. The method for gold analysis is somewhat slow due to the necessity for digestion of the sample prior to analysis, but is a relatively simple procedure. The theoretical minimum concentration of bromine that can be analyzed by this method, taken as the concentration giving a peak which has a net intensity of three times its standard deviation as calculated from counting statistics, is 0.1 mg/100 ml. For iodine the minimum is 0.8 mg/100 ml. Both can be lowered by using longer counting times. However, in the case of bromine, this is not really necessary as the limit is already below the range of bromide levels normally found in serum, estimated as 0.25-2.0 mg/100 ml (as NaBr) (9). Levels above this occur from drug administration. Intoxication occurs a t levels above 150 mg/100 ml and more serious toxicosis above 250 mg/100 ml (10). Samples containing bromide a t levels above 24 mg/100 ml require dilution with water before measurement, and then the sample matrix even more closely approximates that of the standards. The normal levels for iodine in serum are in the range 4.8-8.6 pg/lOO ml ( 1 1 ) which is well below the detectable limit of this system. Ours is not the best fluorescence sys-
tem for iodine measurement. The choice of an Ag target means that the iodine K a line, occurs in the region of highest background intensity. Also, only the higher energy continuous radiation can be used to excite the iodine K a line since the intense Ag K a and KP lines are too low in energy. However, we still find the method useful for measuring levels of iodine-containing compounds in spinal fluids, where they have been used as X-ray contrast media. The gold analysis suffers the disadvantage of requiring digestion before measurement, unlike an earlier atomic absorption method (12). But it is still a relatively simple procedure and, with a detection limit as previously defined of 0.2 pg, is reasonably sensitive and, as can be seen from Table VI, quite accurate. The lower limit of detection is equivalent to 0.05 ppm on a 4-ml sample or 5 pg/lOO ml, which compares very favorably with the atomic absorption study and is well within the clinical range of interest, -100 pg/lOO ml (12). As in the previous study, the accuracy of measurement a t or just above the limit of detection can be improved by addition of known amounts of gold. The standard deviation in a sample containing 1 pg Au is f 0 . 2 pg while for 10 pg, it is f0.25 pg.
LITERATURE CITED (1)F. Claisse, Que. Dep. Mines Preliminary Rep., 402 (1960). (2)E. L. Gunn, Anal. Chem., 33, 921 (1961). (3)Y . Deutsch. Anal. Chem., 46, 437 (1974). (4)H. J. Rose, Jr., and F. Cuttita, Adv. X-Ray Anal., 11, 23 (1968). (5) R. K. Lund and J. C. Mathies, Norelco Rep., 7 , 134 (1960). (6)J. S.Wahlberg and A. T. Myers, U.S. Geol. Surv., Prof. Pap. No. BOO-D, 214 (1968). (7)S.Natelson and B. Shied, Clin. Chem., 8, 17 (1962). (8)C. L. Luke, Anal. Chim. Acta, 41, 237 (1968). (9)G. A. Harrison, "Chemical Methods in Clinical Medicine", J. and A. Churchill Ltd., London, 1957,p 358. (IO)E. Rentoul and H. Smith, "Glaisters Medical Jurisprudence and Toxicology", 13th ed., Churchiii Livingston, Edinburgh and London, 1973,p 558. (11) "Handbook of Biological Data", National Acad. of Sciences, Nat. Res. Council, W. B. Saunders and Co., Philadelphia and London, 1956,p 52. (12)A. Lorber, R. L. Cohen, C. C. Chung, and H. E. Anderson, Arthritis Rheum., 11, 170 (1968).
RECEIVEDfor review March 31, 1975. Accepted June 9, 1975. The authors are grateful to the National Research Council of Canada for partial financial support.
X-Ray Fluorescence Spectrometric Determination of Selenium in Biological Materials K. 1. Strausz Toxicology-Provincial Analyst Laboratory, Alberta Department of Agriculture, Edmonton, Alberta, Canada
J. T. Purdham and 0. P. Strausz Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
Selenium has been shown to be an essential nutrient (1) and deficiencies in animal diets lead to serious diseases. At the same time, selenium levels slightly above the nutritional requirement are highly toxic and the permissible intake level has been estimated to lie in the range 0.5 to 3.5 pg/g of feed (2). The recognition of the biological role of selenium and the critically narrow concentration range between deficiency and toxic levels has given stimulus to the development of new sensitive analytical methods for the determination of this element. 2032
The techniques most commonly used for selenium determination are neutron activation analysis ( 3 ) , fluorometry (4, 5 ) , atomic absorption (6, 7) and spectrophotometry (8). The first method has the obvious disadvantages of being expensive and time-consuming, while the last three often require elaborate, multi-step procedures and are subject to interferences. We wish to report here a rapid and simple method for the analysis of selenium in biological matrices using X-ray fluorescence spectrometry. The technique is not sensitive
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
enough for the direct determination of selenium since typical detection limits are 2-3 ppm, and preconcentration is required. Thus, Wanatabe et al. determined selenium in water by precipitating it as the diethyldithiocarbamate complex (9), a method suggested by Luke (IO). Precipitation with diethyldithiocarbamate, however, is not readily adaptable to the determination of selenium in biological systems because the nonspecific nature of the reagent causes the precipitation of other metals, notably iron, which is present in large quantities in many samples, e.g., blood and liver, preventing the preparation of the sample as a thin film. In the present method, we reduce selenium to its elemental form using tellurium as coprecipitant.
EXPERIMENTAL Apparatus. Intensity measurements were made on a Philips P W 1540 X-ray fluorescence spectrometer. A silver target anode, operated a t 50 kV and 20 mA, was used to produce the exciting radiation, and a lithium fluoride (200) crystal, in conjunction with appropriate pulse height selection to eliminate interference by the second order Ag K a line, was employed for the isolation of the Se KO line. Intensities were measured with a thallium doped sodium iodide scintillation counter. Reagents. 1) Digestion mixture: -3:l HNOs:H*S04. 2) Reducing agent: a stannous chloride/hydroxylamine hydrochloride reagent is prepared as follows: Add to 600 ml water, in order, 100 ml concentrated sulfuric acid, 20 g sodium chloride, 40 g hydroxylamine hydrochloride, and 40 g stannous chloride dihydrate. Cool and filter the solution and adjust the volume to 1000 ml. 3) Standard solutions of selenium with concentrations of 1 ppm and 10 ppm, prepared by dilution of a standard 1000 ppm solution supplied by Harleco. 4) 4 solution containing 100 ppm tellurium, prepared by dissolving 0.125 g TeOz in hydrochloric acid and diluting to 1000 ml. 5) A solution containing 15 mg Cu (as CuSOd/ml. Procedure. The digestions are carried out in screw-cap plastic bottles to prevent any loss of selenium during this procedure (11, 1 2 ) The reducing agent was chosen because it had been demonstrated that, unlike most other reducing agents which can be used only in a hydrochloric acid medium, the present reagent can be employed in nitric acid/sulfuric acid mixtures (13) The procedure for a typical analysis is as follows: 5 g of specimen, together with 10 ml of digestion mixture, are transferred to a plastic screw- cap bottle of dimensions 9 X 4 cm. The bottle, with its cap screwed on tightly, is left to stand overnight and then kept on a water bath a t 60 O C for an additional period of four hours. After cooling, the contents are washed into a 100-ml beaker, diluted to 40-50 ml, covered with a watch glass, and simmered for one hour to drive off the bulk of the oxides of nitrogen. The solution is then filtered, if necessary, and 1 ml of CuSO4 solution (15 mg Cu/ ml), 400 pg of tellurium, and 15 ml of reducing agent are added in order. The solution is allowed to stand for 10-15 minutes. After this time, the precipitate formed is filtered on a 0.45-km-pore Millipore filter membrane and allowed to dry for one hour in the air. The filter disc is placed in a sample holder and a 1.5-mm thick Teflon disc placed over it to keep it flat. The sample is then introduced to the X-ray beam, and counts accumulated for 100 sec a t the position of the Se Kcu line (20 = 31.87O). Background measurements are made for the same time a t 28 = 31.0' and 32.74'. The peak is corrected for background intensity and the selenium content is calculated from the calibration line established as described below.
RESULTS AND DISCUSSION The calibration was achieved by precipitating known amounts of selenium in the 0- to 40-pg range from solutions under conditions identical to those employed for the samples, except that after the addition of the digestion mixture, the standards were not put through the digestion procedure. The calibration curve of net Se K a counts/100 sec vs. pg Se is linear, passes through the origin, and extends to -120,000 counts for 40 pg Se, and is defined by the equation, Y = 3046X -. 101. All seven calibration points (0, 5, 10, 20, 25, 30, and 40 pg Se) lie directly on the line. The linearity indicates that the precipitation of Se is complete. The method was further tested by adding known
Table I. Recovery of Added Selenium from the Digestion-Precipitation Procedure Se added,
ug
Reagent blank 0 .o
0 .5b 1.Ob 2 .Ob 5 .Ob 15.0* 25.0b 0.5' 1.5' 5 .Oc
Se found, ug
Se recovered
d
ug
Recovery, 7'0
0 .o
O.4lu 0.92 1.36 2.48 5.10 14.53 25.33 0.85 1.83 5.05
102 .o 95.0 103.5 93.8 94.1 99.7 88.0 94.7
0.51 0.95 2.07 4.69 14.12 24.92 0.44 1.42 4.64
92.8 Average 96.0 i 4.6% a Average of two determinations. Selenium added as selenite. Selenium added as the diethyldithiocarbamate complex. Amount of Se recovered from the amount added.
I 20
10
mg
Cu a d d e d
Flgure 1. Effect of copper addition on selenium recovery in the presence of 300 pg tellurium
i 1
pg Te a d d e d
Figure 2. Effect of tellurium addition on selenium recovery in the presence of 10 m g copper
amounts of selenium to digestions containing 5 g of skeletal muscle. Selenium was added as selenite and as the diethyldithiocarbamate complex. A reagent blank and two controls containing 5 g skeletal muscle to which no selenium had been added were put through the procedure at the same time. The results obtained are presented in Table I. The recovery in each case was reasonable and averaged 96.0%. The minimum detectable limit, defined as that quantity which gives a peak greater than three times its standard deviation calculated from counting statistics, is 0.2 wg which corresponds to 0.04 ppm in a 5-g sample. This limit can be lowered by using longer counting times when
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
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small amounts of selenium are present. The relative standard deviation on a measurement of 0.5 pg is 20% while for 2 pg, it is 5%. Both copper and tellurium are necessary for the complete precipitation of selenium using the reducing agent described above. Figure l shows the effect of adding increasing amounts of copper to a digested sample of skeletal muscle to which 20 pg selenium and 300 pg of tellurium have been added, and Figure 2 shows the effect of adding tellurium in increasing amounts to digests which contained 20 pug selenium and 10 mg copper. From these two experiments, it was determined that 15 mg copper and 400 pg tellurium are required for complete precipitation of selenium. Other elements, in particular iron, do not interfere with the process since full recovery of selenium is obtained from solutions containing up to 10 mg iron. The method, while not as sensitive perhaps as others such as neutron activation and fluorometry (3, 4 ) does offer the advantage of being extremely simple and, after digestion it is fairly rapid, making it readily adaptable to routine laboratory analyses.
ACKNOWLEDGMENT The authors thank E. M. Lown for reading the manuscript. LITERATURE CITED (1) K. Schwarz and C. M. Foltz, J. Am. Chem. SOC., 79, 3292 (1957). (2)R . A. Passwater, Fiuoresc. News, 7, 1 1 (1973). (3) L. J. McGee and G. G. Boswell, Talanta, 15, 1435 (1968). (4)J. H. Watkinson, Anal. Chem., 38,92 (1966). (5)I. Hoffmann, R . J. Westerby, and M. Hidlroglou, J. Assoc. Offic. Agric. Chem., 51, 1039 (1968). (6)K. C. Thompson and D. R. Thomerson. Analyst (London), 99, 595 (1974). (7)Y. Tamamoto, I. Kumamaru. Y. Hayashi. and M. Kake, Anal. Lett., 5, 717 (1972). (8) K. L. Cheng, Anal. Chem., 28, 1738 (1956). (9) H. Wanatabe, S.Berman, and D. S.Russell, Taianta, 19, 1363 (1972). (10)C.L. Luke, Anal. Chim. Acta, 41, 237 (1968). (11)D. M. Fogg and N. T. Wilkinson, Analyst(London), 81, 525 (1956). (12)T. T. Gorusch, Analyst(London), 84, 135 (1959). (13)W. R. Hatch and W. L. Ott, Anal. Chem., 40, 2085 (1968).
RECEIVEDfor review March 31, 1975. Accepted June 9, 1975. The authors are indebted to the National Research Council of Canada for financial assistance.
Determination of Total Iodine in Milk by X-Ray Fluorescence Spectrometry and Iodide Electrode Eric A. Crecelius Battelle-North west, Richland, Wash. 99352
Radioiodine isotopes are volatile fission products which may be released to the environment from nuclear reactors and fuel element separation plants. There is particular concern about radioiodine in cow’s milk, since the air-grasscow-milk-man pathway is a major route by which man may receive a radiation exposure. When analyzing for radioiodine, total stable iodine must be determined, since the more accurate analyses are based on addition of stable iodine carrier and subsequent yield measurement of the recovered carrier. Studies have shown that essentially all of the iodine in fresh cow’s milk is present as iodide (1, 2 ) . However, when milk is preserved for storage with formaldehyde, some of the iodide changes chemical form and apparently becomes organically bound. Therefore, a method based on measuring ionic iodide cannot be used for total iodine analysis when milk has been preserved with formaldehyde. Milk is often stored with formaldehyde for subsequent analysis ( 3 ) ,and therefore a technique was needed that would determine total iodine. Two new techniques are described in this note for the measurement of iodine in milk. These techniques include the direct X-ray fluorescence analysis of a freeze-dried milk sample and the use of an iodide electrode method for analysis of fresh milk.
EXPERIMENTAL Sample and Standard Preparation. Twenty-five grams of milk were freeze-dried, producing approximately 3 g of milk powder. The powdered milk was pressed under 5000-kg pressure to produce a pellet 3.0 cm in diameter by 3-4 mm thick. Small variations in pellet thickness due to differing amounts of milk solids produced variations in count rate of less than +5%. Standards were prepared by the method of known additions. Aliquots of milk sample were spiked with known amounts of KI, then freeze-dried and pressed. 2034
To check for iodine loss during the freeze-drying process, both fresh milk and milk stored with formaldehyde were spiked with radioactive 1311 as KI before freeze-drying. After freeze-drying, our analysis indicated that 100 f 2% of 1311remained in the dried milk. X-Ray Fluorescence Analysis Method. The iodine in the milk pellet was analyzed for 1-3 hr using a 100-mC 241Am (60 keV) isotopic excitation source (Kevex, Inc., Burlingame, Calif.). The iodine K a l X-rays (28.6 keV) were measured with a Si(Li) diode (80 mm X 5 mm) with 180-eV resolution FWHM a t 6.4 keV (Kevex, Inc.). Data storage and reduction was done by a 1024 channel analyzer and microcomputer (Model 4420, Nuclear Data, Palatine, Ill.). The I Ka count rate was 15 f 1 cpm/ppm I. The background count rate for the I K a peak was 60 f 4 cpm. The only interfering X-ray in the energy region of the I K a l peak was due to tin K/31 X-ray a t 28.5 keV. The system blank caused by traces of tin in the collimator was determined by counting materials of a similar basic matrix but which contained low concentrations of iodine and tin. These materials include cellulose, Plexiglas, sucrose, and high purity water. All of these materials, which were counted at the same total count rate as the milk pellets, produced the same Sn K/31 count rate of 5.8 f 0.5 cpm. This count rate for Sn KP1 was used as a blank and subsequently substrated from all iodine X-ray peak measurements. The possibility of a significant tin content in milk was checked by examining the tin K a l peak (25.3 keV). No observable tin was seen in the milk samples analyzed. Iodide Electrode Analysis Method. Fifty ml of fresh milk or thawed fresh frozen milk is placed in a beaker and 50 mg reagent grade KC1 added to increase the electrical conductivitiy. The iodide concentration is determined by using the “method of addition scale” on an Orion specific ion meter (No. 407A), an Orion iodide electrode (No. 94-53), and an Orion double junction reference electrode (No. 90-02). Reagent grade KI solution was used to make the known additions. The method thus simply involves a direct reading of the ion meter with the sample in place, followed by one or more subsequent readings after known addition of iodide as KI. In our experience, the ion meter scale was found to be linear over the iodide concentration observed in milk. The electrode output ranges from -138 mV for 10 ppm I to -20 mV for 0.1 ppm I. The limit of detection is +10 mV for 0.05 ppm I.
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