Anal. Chem. 1980. 52, 1292-1296
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X-ray Fluorescence Determination of Trace Selenium in Organic and Biological Matrices Sotirios E. Raptis, Wolfhard Wegscheider, and Gunter Knapp i n s f k t fur Analytische Chemie, Mikro- und Radlochemie, Technische Universitat Graz, Technikerstrasse 4, A-80 10 Graz, Austria
Gunther Tolg Max-Planck-institut fur Metallforschung, Institut fur Werkstoffwissenschafn, Laboratorium fur Reinststoffe, D- 7000 Stuttgart und Schwabisch Gmund, West Germany
A method is developed to determine selenium in the ng/g and pg/g range in organic and biological matrices. I t is based on a two-step reduction using 5 N HCI and NaI. The elementary selenium is subsequently deposited on a small membrane filter. The procedure was optlmlzed using ’%e as tracer and systematic errors were eliminated. No interferences were found in the chemical preparation step or in the X-ray fluorescence measurements. The detection limit is 70 ng absolute, corresponding to 140 ng/g for a 0.5-9 sample. The linear range extends through 150 pg Se on the filter. The results were checked using NBS-SRM 1567 Wheat Flour, NBS-SRM 1568 Rice Flour, and IAEA A-6/1975 Fish Solubles; no deviations from the certified values could be detected. I n comparison with the atomic absorption hydride evolution technique, the new method is less sensitive, but more accurate with much better precision.
T h e importance of selenium in biological and organic matrices is well established and lies in its dualistic effect on animals and human beings as both an essential ( I ) and toxic ( 2 ) trace element. T h e distinction between these has been difficult because of t h e lack of reliable and fast analytical methods. T h e atomic absorption method based on the evolution of H2Se, is severely impaired by elements like Ag, Bi, Cu, As, Sb, Sn, Co, Ni, Pt, and Mn ( 3 ) . Studies on biological and organic standard reference materials (SRMs) indicate a very complex interdependence of systematic errors even a t low concentrations of concomitant ions. Unless a sample preparation is introduced prior to the determination, the hydride method gives unreliable results ( 4 ) . I n X-ray fluorescence spectrometry (XRF) the relatively low sensitivity makes a decomposition and preconcentration step mandatory. Selenium has recently been determined in different matrices by coprecipitation ( 5 ) ,cocrystallization with a chelating agent ( 6 ) ,and precipitation exchange (7). T h e present method has been developed t o be very insensitive t o concomitant ions, to give better sensitivity and lower detection limits than previous ones, yet offer reasonable simplicity, ease of automation, and speed.
EXPERIMENTAL Reagents. All materials used were of Analytical Reagent quality. The water was double distilled in a quartz apparatus. The Se stock solution was prepared from 81.6 mg of selenic acid (Fluka, Switzerland) by dissolving in 100 mL of 2.5 N HCl. Dilutions from this solution were prepared every other day. The NaI was of “suprapur” quality (Merck, Darmstadt, Order ~ 6 5 1 9 ) . Argon was 99.99%. 75Seas Se20:- was obtained from Amersham, Great Britain, and 37 kBq (1pCi) was used for each experiment. A mixture of chloric and perchloric acid (Merck, Darmstadt, Order $10741) and nitric acid (65%) was used in the wet decomposition experiments. 0003-2700/80/0352-1292$01.00/0
Filtration Apparatus. All parts of the filtration apparatus (Figure 1) that come into contact with Se were made of polytetrafluoroethylene (PTFE);only the filter holder at the bottom is made of polyethylene (Swinnex SX 0013, Millipore Corp., Bedford, Mass.). Two types of filters were used: Sartorius filters 25 mm in diameter (SM 11607,regenerated cellulose, 0.2-pm pore size) and Millipore filters 13 mm in diameter (GSWP 01300, mixture of cellulose nitrate and cellulose acetate, 0.22-pm pore size). These were placed in the filter holder on top of a paper disk (AR 10 01300, MF SUPPORT PAD, Millipore) to ensure a homogeneous deposition of Se. The reduction was carried out in the same vessel. Argon was used to force the sample solution through the filter. X-ray Fluorescence Analysis. All measurements were done on a Philips/Edax EXAM SIX; this instrument was used in the tube excitation mode using a 50-W Rh tube (Watkins-Johnson) and in the secondary target mode using a 3-kW Au tube in conjunction with a Zr target. A Philips PW 1140 generator was operated at 40 kV and 60 mA. The Si(Li) detector had a resolution of 165 eV a t 5.9 keV. Selenium analysis was made with the K a line at 11.21 keV. Gross integrals over 300 eV were taken for the construction of the calibration curves and the analysis of the unknowns. The samples were mounted on a Spectro-Cup (Somar Laboratories, New York, N.Y.) sandwiched between two sheets of Mylar (5 pm, Somar Laboratories). Analytical Procedure. The decomposition of the sample was accomplished by two independent methods; one was a mechanized wet decomposition in a mixture of chloric acid-perchloric acid and nitric acid (4,8). Frequently, the wet chemical decomposition in open vessels leads to severe losses of Se. The high oxidation potential of chloric acid avoids this loss if the optimal temperature is maintained. Using a continuous wet-ashing apparatus (Fa. Anton Paar, Graz, Austria), these optimal conditions are guaranteed. If the wet decomposition is carried out on a heating plate the following parameters have proven optimal: a Kjeldahl flask made of quartz and containing up to 500 mg of sample with 6 mL of chloric-perchloric acid mixture and 4 mL of nitric acid (65%) added is carefully heated for 1-2 h to avoid an overflow of the mixture by foaming. Afterward the flask is slowly heated to 220 “ C and kept at this temperature until white fumes of perchloric acid begin to appear. The acid residue is 0.5 to 1 mL and mainly consists of perchloric acid. Glass vessels are unsuitable because up to 50% of the Se may be adsorbed on the surface of the vessels. The other decomposition method is based on the incineration of the sample in a stream of oxygen (4,9). With the aid of a mechanized apparatus (“Trace-0-Mat”, Fa. Anton Paar, Graz, Austria), this decomposition that is particularly suited for trace analysis is carried out in a simple manner. About 500-mg samples are pressed into pellets and burned in the quartz apparatus. The inorganic residue from each pellet is collected in 2 mL of HCl (20.2%). The recovery is about 95%. The residue of the wet decomposition is first reduced with 2 mL of 5 N HC1; then 4 mL of water are added and the solution is immediately transferred to the filtration vessel. The decomposition vessels are washed twice with 2.5 mL of H 2 0and these volumes are added to the digestion residue in the filtration vessel. Dissolved oxygen is removed by flushing the solution with Ar for 10 s. T o reduce the selenite to elementary selenium, 1 g of solid NaI is added. After 10 min, the filtration is started and takes about 3 min. The 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980
-M45x2
----I
1293
2 /
D 0001
T
-
01
0.01
-
2
O.!j
5
10
Nai conc in solution ( % I
Flgure 3. Recovery of selenium as a function of NaI content. Sample: Residue from decomposition, 2 mL of 5 N HCI, 8 mL of H,O, 1 p g of Se, 15-min reaction time, 5-min filtration time
Flgure 1. Filtration apparatus (all data in mm, M stands for "metric"). (1) Argon inlet. (2) Screwing cap of stainless steel. (3) PTFE body. (4) Filterholder Swinnex 0013
1 t
- - 07
1
30
+
120
+
4
--__
361
-
lime i m i n l
Figure 4. Recovery of selenium as a function of reaction time. Sample: Residue from decomposition, 2 mL of 5 N HCI, 8 mL of H,O, 1 p g of Se, 1 g of NaI, 3-min filtration time
1;1
t
Table I. Dependence of Recovery on Sample Volume
L
20
volume of sample, m L
0~ 0 001
10
0.0125
0 125
1
-
2
4
10
5 10
HCi normaiity
20 30
Figure 2. Recovery of selenium as a function of acidity. Sample: 5 N HCI, diluted with H,O to 10 mL; 1 wg of Se as SeO,*-, 1 g of NaI, 15-min reaction time, 5-min filtration time
filters are washed with 2 mL of HzO and air dried for 15 min. Afterward the filters are mounted and can be analyzed immediately.
RESULTS AND DISCUSSION Optimization of the Preconcentration Step. The reduction of selenite to elementary selenium by iodide has been used for iodometric (10) and volumetric ( 1 1 ) determinations and can be written as: Se032- + 41-
+ 6H+
-
Seelem+ 21,
+ 3H20
(1)
This reaction yields S e in a red modification. Preliminary studies showing that this reaction and the separation of Seo can be made quantitative led to a radiometric optimization of various parameters, e.g., the concentration of acid in the sample, the amount of iodide, and the time and volume dependence of the reaction. Physical phenomena, like crystal formation and dissolution of the elementary Se, have been studied only in the context of the reaction and filtration. The acid concentration in the sample solution is of importance for the reduction as well as for the dissolution of the formed Se. This dissolution can take place either in the solution or after deposition of the Se on the filter. Too high an acid
40 50 100 a
recovery on the filter,= %, ( N = 4) 98.1
i
1.3
98.4 98.3 98.2 97.1 94.2
i
1.5 1.1 1.4
i
*
i i
1.2
1.5
9 0 . 5 ? 1.3
100 ngof Se.
concentration would also lead to a swelling of the filter. The optimal acid concentration is between 0.125 and 1 N HC1 (Figure 2), but since an excess of acidity is required, 1 N HC1 was used. Figure 3 gives the dependence of the recovery on the iodide concentration. Quantitative reduction could be achieved only for a NaI concentration of more than 1%.The use of strongly oxidizing acids (HN03,HC103, HC104) in the decomposition step suggests the operation a t the upper end of the curve to ensure sufficient excess of iodide; the addition of 1g of NaI in solid form prevented the potential formation of products from redox reactions in a NaI solution and gave a final concentration of about 10%. Consumption of iodide by dissolved oxygen could be prevented by purging the solution with Ar for 10 s, through a capillary tube. T h e time for the reaction prior to filtration has to be sufficiently long not just to complete the reduction but also for the formation of sufficiently large particles for filtration. The latter step takes longer than the reduction alone. Figure 4 shows that a t least 10 min are required for the crystallization.
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980
Table 11. Influence of Filter Material and XRF Excitation Conditions filter diameter, mm 25 13 13
excitation conditionsa A
supplier Sartorius Millipore Millipore
A
B
detection limit, ng for 1000-s counting time 470 40
RSD at 1 Pg, Yo 19 1.2 2.3
70C
sensitivity, WSIP g
1.3 2.1
5.4
a A: Rh tube in pulsed mode, 35 kV, 990 PA 0.125 mm Rh filter. B: Au tube, secondary target mode, target material: Se added prior to decomposition. Se adled after decomposition. Zr, 40 kV, 60 mA.
Table 111. Amounts of Concomitant Elements Used in Interference Experiments
ion Co2+,Ni", Pb",
Mn4+,Cu2+ As5', Sb5+,Ag', Bi3+ Sn4+,Cd2' Mg", BaZ+,Srz+ Znz+ Fe3+,CaZt K + , Na+ total
corresponding excess over Se concn. in a weight ratios 0.5-g sample, to Se Pglg 50 each 100 each 100 2 00 250 500 5000
10000
12450
24900
200
400 500 1000
After 2 h, the dissolution of Se begins. The dissolution is also volume-dependent b u t the separation is quantitative below 40 m L (Table I). T h e reduced recovery a t higher volumes is, however, very reproducible and a determination of trace Se in water therefore seems possible. T o avoid lengthy filtration, the optimal sample volume is between 10 and 15 mL. T h e separation is quantitative between 10 ng and 200 pg Se. As adsorption effects of Se03'- are well known (12),it was of interest to study losses of Seo on different materials: At the 100 ng level of Se, 7.1% is adsorbed on polystyrol, 16.8% on glass, 1.1%on PTFE, and 1.3% on quartz. Since machining is easier with PTFE, this material was used for the filtration equipment, while quartz vessels are used in the decomposition apparatus. The filtration time was varied between 3 and 15 min and had no influence on the recovery or the homogeneity of the deposit. With an Ar pressure of 3 N/cm2 the filtration was complete in 3-4 min. T h e size and the acid stability of the filters had a marked influence on the performance characteristics of the method. The poor precision for the 25-mm Sartorius filter is attributed to two facts: (1)the lower stability in the acidic medium led to shrinking and wrinkling of the filters; (2) it is possible that the larger diameter also increased the inhomogeneity of the precipitate. T h e Millipore fi!ters used are reported to have a blank value of 0.4 ng of Se (13), well below the detection limit of the present method. The calculation of the detection
limit was carried out according to Currie ( 1 4 ) ; the general assumption of uo = u,that is the variation of the background is equal to the variation of the lowest standards is not valid in the case of preconcentration work and leads to unrealistically low figures. Thus, separate evaluations of these two quantities were used in the calculation of the detection limits to accurately reflect the performance of the method a t very low levels (Table 11). Interference Studies. No interferences could be detected radiometrically for levels of concomitant ions that exhibit a marked influence in the hydride generation technique. This was expected as the reduction of Se is very selective and an 85000-fold excess of iodide is used. T o study interference effects in XRF-measurements, two sets of Se samples containing 1 pg each were prepared: one set was treated like the standards while large amounts of potential interferents were added to the second set. The total excess over Se was more than 12000-fold, about 5-10 times more than can reasonably be expected in biological and organic samples (Table 111). Gross intensities were 1.4% low in this experiment because of reduced background only when the washing step was omitted. No reduction of recovery was found. Determination of Se i n S t a n d a r d Reference Materials. A calibration curve was constructed and the linear range extends through 150 pg. The determination of Se in standard reference materials was accomplished with a calibration curve where Se was carried through the entire procedure including the decomposition to account for the 98.5% recovery determined before. Alternatively, a correction factor can be employed. The stability of the standards mounted on SpectroCups and covered by Mylar has been checked 4 months after preparation and no changes in accuracy and precision could be detected. Dissolution of the filters in a small volume of concentrated H N 0 3 makes this preparation method useful for accurate atomic absorption measurements either by flame or graphite furnace atomization. Se could easily be quantified in all three reference materials (Figure 5); a summary of the results is given in Table IV. Comparison to the Hydride Evolution Technique. The present method is inherently less sensitive than the AA method after evolution of the hydrides. However, the much lower detection limit (2.5 ng vs. 70 ng), does not reflect upon the
Table IV. Results of Reference Analyses This Method
sample
certified, @gig
wet chemical decomposition sample result, weight, mg Pg/g 500 0.4 t 0.1
NBS-SRM 1568 Rice Flour
0.4
NBS-SRM 1567 Wheat Flour
1.12 0.2=
350
1.0
f
O.lb
IAEA A-611975
3.07
300
3.1
t
O.lb
f
0.1'
t
0.6b
incineration result , weight, mg PgIg 500 0.40 r 0.02b sample
1.10 * 0.02b
500
Fish Solubles a
Total expected error.
95% confidence limit.
-
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___
ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980
A
1295
E3
c
Figure 5. Spectra of filters after preconcentration of Se from SRMs, Se Kcu located at vertical line. (A) NBS-SRM 1568 Rice Flour (-0.2 pg of Se). (B) IAEA A-6/1975 Fish Solubles ( - 0 9 2 pg of Se). (C) 0 25 pg of Se standard. (D) NBS-SRM 1567 Wheat Flour (-0.36 c(g of Se)
_______-
Table V. Comparison of the XRF Method and the AA Hydride Evolution Method
sample
method
International Atomic Energy Agency A-611975 Fish Solubles
AA hydride evolution
National Bureau of Standards 1 5 6 8 Rice Flour National Bureau of Standards 1567 Wheat Flour a
sample size, mg
detection limit in sample.
RSD
interference
(N= 4), from sample, 5%
%a
8
0.30
6.1
-15
this method, wet decompos./XRF
300
0.13
1 6
n.sSb
AA hydride evolution
120
0.02
2.7
-
this method, incineration/XRF AA hydride evolution
500
0 08
0.3
ns.
40
0.06
4.9
-29
this method, incineration/XRF
500
0.GS
0.7
ns.
Relative difference of certified value and found value.
detection limits in the original sample because the sample size cannot be increased at will: judging from the matrix effects the upper limit is reached for the hydride method (Table V).
Pg/g
10
n.s. stands for “not significant”.
For the XRF method, the sample sizes given me not dictated by interference effects, but constitute upper limits imposed by the decomposition methods. It is therefore reasonable to
Anal. Chem. 1980, 52, 1296-1300
1296
expect even lower limits as the decomposition methods are developed to manage higher initial sample sizes. The great reliability and the relative simplicity makes this a competitive technique among currently used methods for the determination of selenium.
ACKNOWLEDGMENT The authors appreciate the help of E. Rubner who built the filtration apparatus.
(5) Vassilaros, G. L. Talanta 1971, 18, 1057-59. (6) Lindner, H. R.; Seltner, H. D.; Schreiber. B. Anal. Chem. 1978, 50, 896-97. (7) Disam, A.; Tschopel, R.;Toig, G. Fresenius’ 2.Anal. Chem. 1979, 274, 97- 109. (8) Knapp, G. Fresenius’ Z . Anal. Chem. 1975, 274, 271-73. (9) Knapp. G.; Kaiser, G.; Toig, G.; Schreiber, B. Technical University of Graz, Austria, 1979, unpublished work. (10) Geiersberger, K. Fresenius’ 2. Anal. Chem. 1952, 135, 15-18. (’ ’) Kainz, G.; Resch, A. Mlkrochem. 1953, 40,332-42. (12) Shendrikar, A. D.; West, P. W. Anal. Chim. Acta 1975, 74, 189-91. (13) Mc Donald, C.; Duncan, H. J. Anal. Chim. Acta 1978, 102, 241-44. (14) Currie, L. A. Anal. Chem. 1968, 40,586-93.
LITERATURE CITED Schwartz, K.; F o W c. M., J . Am. Chem. S O C . 1957, 79,3292-97. Underwood, G. J. “Trace Elements in Human and Animal Nutrition”, 3rd ed; Academic Press: London-New York, 1971. M e w , A.; Hofer, Ch.; Tolg, G.; Raptis, S.; Knapp, G. Fresenius’ Z. Anal. Chem. 1979, 296, 337-44. Raptis, S.;Knapp, G.; Meyer, A,: Tolg, G. Fresenius’ z. Anal. Chem. 1980, 300, 18-21.
RECEIVED for review J~~~~~~~ 14,1980, ~
~ ~~~~h ~ 26, 1980. This work was supported in part by grant ~ 3 5 4 3from the ‘‘Fonds zur Fbrderung der wissenschaftlichen Forschung”, Vienna* Additional support was provided by Sandoz AG, Switzerland.
Polarization Ratio of a Diffracted-Beam Monochromator in X-ray Powder Diffractometry Stephen Altree-Williams Division of Occupational Health and Radiation Control, Health Commission of New South Wales, P.O. Box 163, Lidcombe, Australia 2 1 4 1
Bernard Jordan Division of Chemical and Physical Sciences, Deakin University, Geelong, Australia 32 17
The polarization ratio Kof a diffracted-beam monochromator may be determined using ratios of diffraction intensities obtained from conventional powder diffractometer arrangements with and without a monochromator. The method allows K t o be conveniently determined for a laboratory’s specific monochromator crystal and diff ractometer arrangement. For the graphite crystal with our diffractometer, the method gave K = 0.75 f 0.04 using chromium radiation and K = 0.93 f 0.04 using copper radiation.
One of the uncertainties in applying theoretically calculated data to quantitative X-ray powder diffractometry is in calculating the effect on diffraction intensity due to any monochromator used. In calculating the integrated intensity of a diffraction line ( I , 2) on a conventional Bragg-Rrentano parafocusing diffractometer (3)with an unpolarized X-ray source and without a monochromator, the polarization term, P , is given by
P = yZ(1
+ cos* 245)
(1) where Old is the Bragg angle of the measured diffraction line i of phase J. When a diffracted-beam monochromator is used in the equatorial plane of the diffractometer, as is the conventional configuration (3, 41, the polarization term for the overall system, P’, becomes
P’ =
y2(l + K
cos2 20L5)
cos’ 20M, where OM is the Bragg angle of the monochromator crystal for the wavelength used. Recent theorists ( 5 , 8-1 I) point out that this assumption is not necessarily valid and suggest that the value of K could lie anywhere between cos2 28, and 1, depending on the properties of the specific monochromator crystal used and on the diffractometer geometry (including beam divergence and receiving slit size in the case of the diffracted-beam monochromator). Experimental measurements of K for a number of incident-beam monochromators have given values between cos2 2OM and 1 (9, 10, 1.2). The “0--P” method used for these determinations ( 5 , I Z ) , although capable of very high precision, requires significant modification to the usual diffractometer arrangement; for example, remounting the detector to receive rays normal to the plane of the diffractometer. An alternative, convenient way of assessing the value of K for the diffracted-beam monochromator typically used in quantitative X-ray powder diffractometry is suggested here. The method involves measuring the relative intensity of pairs of diffraction lines of a phase both with and without the monochromator attached to the diffractometer. These relative intensity measurements can then be used to directly calculate K , as follows. Consider the conventional diffractometer without monochromator and the general case of a sample of pure phase intersecting the whole X-ray beam. The integrated intensity of two lines i and j of the phase measured under the same diffractometry conditions is given by ( I , 2)
(3)
(2)
where K is the polarization ratio ( 5 ) of the monochromator for the diffractometry conditions used. Early theorists (6, 7) assumed the monochromator crystal to be ideally mosaic and suggested the value of K would be 0003-2700/80/0352-1296$01 .OO/O
and (4)
where C is a constant dependent on the specific diffract,ometry 1980 American Chemical Society
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