I
$Jy\ ,I a 2 0
J LL
200
300
400 500 600 WAVELENGTH ( n m )
700
Figure 4. Spectra of 5AF-nitrite system. (A) Excitation spectra of 10-7M 5AF; ( B )Emission spectra of 10-7M 5AF; ( C ) Amplification of A; (0) Amplification of lO-'M 5AF, 10-7M NO*-
to be quite complicated. However, they both show excitation-emission at 490-515 nm. These two wavelengths (for the instrument and lamp used in this work) provide the best analytical sensitivity. Figure 4 shows the spectra for the 5AF-NO2- system. Lines A and B show that the 490515 nm combination is best. In addition, excitation at lower
wavelengths can be used but a t reduced analysis sensitivity. While the dye and the dye-nitrite spectra have large excitation maxima a t 490 nm, the spectra differ in the 250400 nm region. Lines C and D are amplifications of that lower nm region. It should be noted that excitation at wavelengths in the 250-400 nm region will produce emissions a t 365-385, 410-425, 690, and 750 nm in addition to 515 nm. However, only the 515-nm peak is suitable for analysis work, as the intensity of the other emission peaks is not altered by the reaction of nitrite with 5AF. It is also possible that some of the non-usable peaks are due to the presence of impurities in the 5AF dye.
LITERATURE CITED (1) C. A. Streuli and P. R. Averell, "The Analytical Chemistry of Nitrogen and Its Compounds", Wiley-lnterscience, New York. NY, 1970. (2) J. ti. Wiersma, Anal. Letts., 3, 123 (1970). (3) L. J. Dombrowski and E. J. Pratt, Anal. Chem.,44, 2268 (1972). (4) H. D. Axelrcd, J. E. Bonelli, and J. P. Lodge, Jr., Anal. Chem., 42, 512 (1970). (5) J. D. Roberts and M. C. Caserio, "Basic Principles of Organic Chemistry", W. A. Benjamin, New York. NY, 1965.
RECEIVEDfor review September 16, 1974. Accepted January 2,1975. The National Center for Atmospheric Research is sponsored by the National Science Foundation.
Analysis of Urine for Trace Elements by Energy Dispersive X-Ray Fluorescence Spectrometry with a Pre-Concentrating Chelating Resin Madhulika Agarwal,' Roy B. Bennett,2 1. G. Stump, and John M. D'Auria3 Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada
The role of trace elements in the body is an area which continues to receive a great deal of attention ( I ) , especially as newer analytical techniques are developed. Various studies have indicated correlations between particular elemental fluctuations and specific medical disorders (2, 3 ) . Analyses of human urine as a diagnostic tool has not been fully utilized, yet urine is easily obtainable, can be handled without complicated apparatus, and contains various trace elements indicative of medical disorder. Diagnostic elements of particular importance recently are copper ( 4 , 5 ) , zinc (5-7), and lead (8) with respect to a variety of medical disorders. Standard instrumental methods of analysis of trace elements include atomic absorption (9) and stripping voltametry (10). Wavelength dispersive X-ray fluorescence spectroscopy of nanogram amounts of chromium in urine has been performed by Beyermann et al. ( 1 1 ) . The advent of energy dispersive X-ray fluorescence spectroscopy (12, 13) does allow multielemental determinations, rapidly and accurately without destroying the samples. The areas of applicability of this new analytical technique are only now Present address, McMaster University Medical School, Hamilton, Ontario. * Present address, Bio-Tracers Services, 1908 Mahon Avenue, North Vancouver, B.C. Author to whom reprint requests should be sent. 924
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
being fully appreciated and it is conceivable that it can provide valuable diagnostic information rapidly when used routinely on urine samples. It should be noted that matrix effects (14) must be taken into account when performing quantitative analysis; however, these could be avoided by using the sample material itself in the analysis. The purpose of the present study was to explore the applicability of an energy dispersive X-ray fluorescence spectroscopy (EDXS) system to determinations of trace elements, e.g., Cu, Zn, and P b in urine. The system available was a photon excitation, secondary target EDXS system (15) and two major factors had to be investigated: a sample preparation scheme which allows determination of the trace elements in the concentration levels normally present in urine; and a sample preparation scheme which is reasonably rapid and efficient but allows for reproducible and quantitative determinations. Various sampling procedures were studied including spotting onto filter paper, freeze-drying urine samples, and using an ion exchange chelating resin, Chelex 100 to provide pre-concentration prior to analysis. Earlier studies by Blount e t al. ( 1 6 ) have utilized similar chelating preparatory procedures for barium ions in an aqueous medium prior to wavelength dispersive X-ray spectroscopy. This resin shows a high preference for divalent and polyvalent metal ions, such as Cu, Hg, Zn, and Pb, over lighter metals corre-
MO
-zz7
( a)
IO['
Y
YI
_J
w
z z a
I
t t
CHANNEL
Figure l(a). An X-ray spectrum of a resin sample treated, as described in the text, with a human urine and water solution, doped with 3.4 ppm solution of Yt ions. The asterisk indicates the peaks which contain some contributions from background. (b) An X-ray spectrum of urine solution with 3400 ppm of Yt ions spotted onto filter paper as described in the text
sponding to the reactivity of iminodiacetic acid functional groups in the resin (17).
EXPERIMENTAL Apparatus. The photon excitation, secondary target energy dispersive X-ray fluorescence system was obtained from the Kevex Corp. and included a Siemens X-ray tube with a gold anode target to act as a source of primary radiation. This primary radiation can fluoresce a secondary target of choice, which was Mo in the present study. The secondary radiation (Mo K a and KP X-rays) can fluoresce sample material with subsequent emitted X-rays detected with a Si(Li) spectrometer. Further details of this system including methods of data acquisition and computer analysis of the resultant X-ray spectra can be found elsewhere (18). With this choice of secondary target and depending upon the level of concentration, elements from about S to Zr can be excited through K-shell vacancies while heavy elements through L-shell vacancies. T h e anode voltage and current in the primary tube were maintained a t 40 kV and 50 mA, respectively. Reagents. Standard solutions of metal salts were prepared from acetates to avoid introducing elements in the sensitive range of the system. Further, lead bound by a chloride could not be exchanged effectively with the resin. Urine samples taken from the same individual were used as soon as possible for the preparation of the standard response functions to minimize precipitation; the same sample was used to prepare the response for each element studied. The chelating ion-exchange resin Chelex-100 (Bio-Rad Laboratories) was 100-200 mesh and activated by treating with 1N sodium hydroxide (ratio 1:3) for 30 minutes in a rotating mixer. The resin was then washed with distilled water. The urine spotting experiments were performed with Whatman No. 42 ashless paper, Procedure. Normally, standard solutions of known composition
Y __
IO' I
C HANrd EL Figure 2(a). An X-ray spectrum of resin sample treated with a urine solution with 3.4 ppm of Yt ions and 50 ppb of Pb ions. See caption for Figure l(a) (b) An X-ray spectrum of an untreated resin sample
were prepared in the ratio of 8:l:l with urine, aqueous yttrium nitrate solution (3.4 ppm), and aqueous solution of element of interest. The yttrium was included for purposes of normalization to take into account instrumental fluctuations, sample preparation variations, and differing sample geometries. Spotting of urine solution onto filter paper for analysis were performed using a 25-rl pipet. A single drop formed a 1-cm2spot size. The procedure for use of the Chelex 100 resin was as follows: 0.1 gram of dried resin, following treatment described above, was mixed with 5 ml of the standard solution for l'h hours on a slowly rotating mixer. The pH of the solution was approximately 7.0. The resin solution was then filtered, dried, and a portion placed in a sample holder for analysis. A similar procedure was followed for studies of ions in an aqueous medium without urine. The EDXS chamber was evacuated and the sample analyzed for periods ranging from 10 to 40 minutes, depending upon the concentration of the element of interest.
RESULTS Typical X-ray spectra obtained from similar solutions containing only urine and yttrium ( a ) soaked with resin as described and ( b ) spotted onto filter paper are displayed in Figure 1. Figure 2a displays the spectra obtained for a urine and yttrium solution with 50 ppb of lead added, again using the resin technique. The identification of each peak was based upon its energies and the expected intensity response for the Ka-KO lines or the La-Lp-Ly lines. It should be noted that the spectrometer did exhibit background peaks for Fe, Ni, Cu, and Zn as displayed in Figure 2b. Table I presents the results for solutions treated with increasing amounts of Zn, Cu, and Pb. The responses are indicated in terms of the area of the X-ray peak of interest ANALYTICAL CHEMISTRY, VOL. 47, NO. 6 , MAY 1975
925
Table I. Results for Solutions Treated with Increasing Amounts of Cu, Zn, and Pb Area of peak/ Concn of final Element of interest
solution,
Area of K u ( o r Lu)peak (average)
Area of Yttrium
Q peak, %
u g / l O O mla
cu
N+ 0 N + 58.4 N
-+
A'
+ 292.0
73.0
N + 146.0 a = 4.161
Zn
b = 0.439
0 + 33.4 + 66.8 + 106.7 + 144.3 + 162.5 + 333.9
N +
N N
N A' 2%'
Pb
0
A'+ A' 'V
+ +
N N AV
A' N
5.1 10.2 + 15.3 25.6 + 51.2 + 102.3 + 204.5
cz = - 0.64
Pb (aqueous solution without urine)
3.7 4.7 18.6 37.2 46.5 92.9 126.8 464.7 a =
0.78
...
402 (30) 400 (20) 537 (20) 1405 (20) 1064 1786 3336
0.7 1 .o 1.7 3.6 6.5 14.2 31.1
Std dev = 2.55
... ...
< 100 < 100 162 (20) 285 467 612 1110 2230 6850 b = 0.044
4.1 9.6 15.0 23.7 28.6 28.9 75.3
0.3 1.2 2.3 2.8 5.4 8.6 20.5
Std dev = 1.36
a N refers to the amount of the element indicated in the urine used while the value reflects the amount added as described in the text. The concentration of yttrium in the final solution was maintained at 3.4 ppm (340 rg/100 ml) and the area of the yttrium K, peak was adjusted to 100. CUnless indicated by a value in parentheses, these areas were for sample count times of 10 minutes. These were determined by a least squares analysis of the data and are the parameters for a linear relationship (y = bn + a ) .
divided by the area of X-ray ( K a ) peak of yttrium. These same data are displayed in Figure 3 where the solid line represents a least squares analysis to the data. On the same figure is displayed the response when an aqueous lead solution, only without urine was studied.
DISCUSSION A comparison of the results between the spotting technique and the resin pre-concentration technique indicated that only the latter provided urine samples of such concentration that the EDXS system could detect above background normal elemental levels (Table I). With the former spotting technique, even multiple spotting did not effect a 926
0
0 ~
10
IW I30 200 ZM 3W 330 COKENTRATION OF URINE SOLUTION WITH ADDED ELEMENT ( y ( 1 1 0 0 m l l
400
Std dev = 3.78
...
b = 0.1527 0
Std dev = 6.84
715 (20) 3705 (20) 8280 (20) 11300 (20) 8003 9810 10500 b = 0.211
a = 0.94
10.4 33.3 26.8 67.1 135.5
lllOC 1640 1115 4045 9760
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6 , MAY 1975
CCWENTRATWN OF SOLUTION WITH LEA0 LDDED 1 ,(/IO0 rnll
Figure 3. The area of the elemental X-ray peak of interest divided by the area of the Ka peak from Yt as a function of concentration of the urine solution with added element of interest in Ig/lOO ml is displayed in (a).In (b)a comparison of the normalized (to Ka of Yt) response for the Pb L a X-ray is indicated between a urine solution and a solution without urine
necessary concentration for detection. The elements observed by the EDXS system using the resin were Cu, Zn, Br, Rb, Sr, and Pb. Normal concentration levels of these in urine range from about 50 ppb to 1ppm ( I , 2 ) . In addition, light elements such as K, C1, and Ca were definitely observed. Finally preliminary studies of urine samples treated with sulfuric acid prior to treatment with resin indicated that V and Cr could be effectively exchanged and detected. The linearity of the normalized peak areas vs. concentration (Figure 3) indicates that for Zn, Cu, and Pb, and probably all of the other elements, the pre-concentrating resin treatment combined with the EDXS system can yield reproducible and quantitative results. The y-intercept reflects the normal level of the element of interest in the urine, and for Cu and Zn, machine background. The differences in slope for the two lead solutions in Figure 3 probably reflects the cation-anion interaction of the resin a t different pH's. The aqueous solution had a p H of 7 while the pH of the resin was usually lower. The normal range for human urine is 6.0 f 1.8 (29).Although it was interesting to note the different response of the resin to the different lead solutions, it was not necessary to fully comprehend the chemical interaction to obtain usable standard curves. An attempt was also made to study Hg in urine samples. While levels in the range of 50 ppb of Hg in an aqueous medium were clearly observable, using the resin technique, Hg added to urine could not be observed. This is attributed t o possible complexes between the species in the urine and Hg, thus effectively reducing the amount of Hg chelated by the resin. CONCLUSIONS The application of the EDXS system to determining quantitatively trace quantities (-1 ppm and lower) of biologically important elements, such as Zn, Cu, and Pb, ap-
pears quite promising, especially as coupled with a preconcentration step using Chelex 100. Any problems associated with the matrix such as enhancement on absorption effects seem to have been corrected by the uniformity of the sample and the normalization process (with yttrium) for the elemental concentration levels normally expected for urine.
ACKNOWLEDGMENT The assistance of D. Appelgarth and G. Davidson of the Children's Hospital in Vancouver in handling biological samples is gratefully acknowledged. LITERATURE CITED (1) H. A. Schroeder and A. P. Nason, Clin. Chem., 17, 461 (1971). (2) E. J. Underwood, "Trace Elements in Human and Animal Nutrition," 3rd ed., Academic Press, New York, NY, 1971. (3) W. Mertz and W. E. Cornatzer, "Newer Trace Elements in Nutrition," Marcel Dekker. New York, NY, 1971. (4) R. W. Vilter, R. C. Bozean, E. V. Hess, D. C. Zellner. and H. G. Petering, NewEngl. J. Med., 188 (1974).
(5)M. Hrgovcic, Progr. Clln. Chem., V, 121 (1973). (6) H. H. Sandstead, Am. J. Clh. Nutr., 26, 1251 (1973). (7) J. Cecil Smith, Jr.. E. G. McDaniel, F. F. Fan, and J. A. Halsted, Science, 181, 954 (1973). (8) J. Jullan Chisolm, Jr.. Sci. Am., 224, 15 (1971). (9) F. W. Sunderman, Hum. Pathoi., 21, 549 (1973). (10) B. Searle, W. Chan, and B. Dovidow. Clin. Chem., 19, 76 (1973). (11) K. Beyermann, H. J. Rose, Jr. and R. Christian, Anal. Chim. Acta. 45, 51 (1969). (12) R. D. Giarque. F. W. Goulding, J. M. Jaklevic. and R. W. Pehl, Anal. Chem., 45, 671 (1973). (13) F. S. Goulding and J. M. Jaklevic, Ann. Rev. Nucl. Sci., 23, 45 (1973). (14) 8.P. Bertin, "Principles and Practices of X-ray Spectrometric Analysis." Plenum Press, New York. NY, 1970. (15) D. E. Porter and R. Woldseth, Anal. Chem., 45, 605A (1973). (16) C. W. Blount. W. R. Morgan, and D. E. Leyden, Anal. Chim. Acta. 53, 466 (1971). (17) D. E. Leydon, Adv. X-RayAnal.. 17, 293 (1974). (18) R. B. Bennett and J. M. D'Auria. hf.J. Appl. Radiat. Isof., 25, 361 (1974). (19) N. W. Tietz, "Fundamentals of Clinical Chemistry," W. B. Saunders. Toronto, Canada, 1970.
RECEIVEDfor review November 15, 1974. Accepted January 6, 1975. Work supported in part with funds from the National Research Council of Canada.
Determination of a-Quartz in Atmospheric Dust: A Comparison between Infrared Spectrometry and X-Ray Diffraction Techniques Alessandro Mangla lstituto di Chimica Generale ed Inorganica, Universita di Parma, 43 100 Parma, Italy
With reference to the problem of the determination of a-quartz in atmospheric dust, the results of a comparison between IR spectrometry and X-ray diffraction are reported. This comparison has been made with the aim of verifying the possibility of the use of IR spectrometry instead of the more expensive X-ray diffraction technique. IR determinations of quartz have already been reported by several authors ( 1 4 , even in the atmospheric dust (3, 4 ) , and the problems of the effects of the particle size and matrix interferences have been widely discussed (1-3, 6). The most commonly-used method involves the removing of the dust from the filter and the determination of the quartz by means of the KBr technique; nevertheless some authors ( 4 ) investigated the possibility of determining the a-quartz content of the dust directly on the filtering membrane. These authors propose to fold the filters to increase the ratio between the amount of dust and the surface of the filter and to reduce the consequences of the inhomogeneity of the dust distribution. In the present work, both the KBr and the membrane technique have been used but, in the second procedure, to improve the sensitivity of the method and to reduce the effects of the inhomogeneity, the samples were transferred from the original membrane to another one, on a surface of the same shape and dimensions of the spectrophotometric entrance port. The results of the two IR procedures have been checked by means of X-ray diffraction on the same sample of dust.
EXPERIMENTAL Apparatus a n d Reagents. Quartz: particle size, 2-10 pm. KBr: mean particle size, 60 pm. Filtering membrane: cellulose nitrate, Sartorius 113.04.050. IR spectrophotometer: Perkin-Elmer model 457. Powder diffractometer: Philips PW 1050, CuKa radiation.
Procedure. The atmospheric dust was collected near a clay factory on a 50-mm diameter filtering membrane. A preliminary investigation of a sample of the dust by means of powder X-ray diffraction showed that only the a-quartz modification of Si02 was present. The samples of pure a-quartz of known particle size were obtained with the sedimentation method, using the Stokes formula to calculate the settling time (7). For the KBr technique, the normal procedure of preparing the pellet was followed and KBr of 60pm mean particle size was used. The spectrum was run three times, with a rotation of the pellet through 60" each time, and the resulting absorbances were averaged. In preparing the standards of quartz on the membrane, weighted amounts were transferred with water on 13-mm diameter filtering membranes using a Plexiglas support (Figure 11, with the filtering surface of the same shape and dimension as the spectrophotometric entrance port (the internal surface of the funnel was accurately smooth to avoid any deposit of dust particles). The quantitative transfer of the substance was controlled by means of a microscope; no loss resulted. The discs were then dried and scanned using the normal sample holder for the 13-mm pellets. A second membrane disc was used for reference; both sample and reference discs were cut from the same filtering membrane in a region close to its center (to avoid the effects of a possible difference in thickness). The same procedure was followed for the unknown samples, after ignition of the original filters carrying the collected dust, in platinum crucibles a t 500 "C. For the X-ray analysis, the standards were prepared adding to the weighted quartz, CaC03 up to 2 mg when their weights were less. The same procedure was followed for the unknown samples after their ignition a t 500 "C. Then, they were transferred onto the membranes, using the previously described support but with the filtering surface equal to the irradiated zone a t 20 = 26.70'. The membranes were then placed on the holders. In this way, strictly flat and homogeneous samples were obtained. For the direct comparison of the methods, a suitable amount of atmospheric dust was collected on a filter and, after burning in a platinum crucible at 500 OC, three portions of it were used for the two IR methods and for the X-ray control. ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
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