Anal. Chem. 1994,66, 3624-3631
Determination of Total Chromium in Whole Blood, Blood Components, Bone, and Urine by Fast Furnace Program Electrothermal Atomization AAS and Using neither Analyte Isoformation nor Background Correction Victor A. Granadlllo, L l e h Parra de Machado,t and Romer A. Romero'v*
Laboratorio de Instrumentacibn Anahtica, Facultad Experimental de Ciencias, La Universidad del Zulia, Maracaibo, Venezuela
Fast furnace program (total furnace time - 100 samples). Fast furnace programs for Cr (total furnace time 150 firings before test); (c) new uncoated graphite tubes; and (d) new pyrolytic graphite-coated graphite tubes with standard pyrolytic graphite L’vov platforms. The ETA-AAS absorbance-time profiles and appearance times for aqueous Cr (15 pg/L Cr, equivalent to 150 pg in the 10-pL injection volume),
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Table 3. Comparlson of Integrated Absorbance Readings. Obtalned for the Fast Furnace Program ETA-AAS Determlnatlon of Chromlum In Whole Blood, Red Blood Cells, Urlne, and Minerallzed Bone Speclmens, Uslng the DABC and ZEBC Instrumental Derignr, wlth and wlthout Background Correctlonb
integrated absorbance (s) DABCd sample
ZEBCd
Cr concnC(pg/L)
ON'
OFF'
ON'
OFFC
0.50 1.60 2.40
0.008 f 0.0003 0.025 f 0.0008 0.035 f 0.0020
0.008 f 0.0010 0.026 f 0.0021 0.035 f 0.0027
0.006 f 0.0002 0.020 A 0.0012 0.029 f 0.0012
0.006 f 0.0003 0.019 f 0.0009 0.028 f 0.0022
1.02 1.86 5.80
0.016 f 0.0009 0.029 f 0.001 1 0.090 f 0.0012
0.016 f 0.0010 0.030 f 0.0022 0.091 f 0.0015
0.012 f 0.0003 0.022 f 0.0015 0.070 f 0.0028
0.013 f 0.0005 0.023 f 0.0010 0.069 0.0022
0.56 1.34 2.70
0.009 f 0.0005 0.021 f 0.0018 0.042 f 0.0023
0.008 f 0.0002 0.019 f 0.0023 0.045 f 0.0016
0.006 f 0.0003 0.017 f 0.0010 0.032 f 0.0031
0.007 f 0.0008 0.016 f 0.0010 0.032 f 0.0025
1.80 4.50 5.48
0.028 f 0.0018 0.070 f 0.0010 0.085 f 0.0005
0.027 f 0.0012 0.069 f 0.0050 0.083 f 0.0038
0.021 f 0.0009 0.056 f 0.0030 0.064 f 0.0008
0.022 f 0.0010 0.054 f 0.0014 0.066 f 0.0035
whole blood 1 2 3
red blood cells 1 2 3
urine 1 2 3
mineralized bone 1 2 3
,IMean f one standard deviation of 10 samples. No analytical isoformer used. In diluted test portions. For each sample analyzed, integrated absorbances obtained with the DABC design were significantly different (p C 0.01) from those of the ZEBC design. For each sample analyzed, no significant differences (p > 0.01) were observed within the same instrumental arrangement, whether or not background correction was used.
n
I\
1.15 s
/I
1.15s
14
0
5
0
5
0
5
0
5
Time (s) Figure 2. Absorbance-time profiles with the cwesponding appearance times (5) for Cr determined by ETA-AAS in the DABC design, using (1) new pyrolytic graphitacoated graphite tubes (no firing before test), (2) used pyrolytic graphite-coated graphite tubes (>150 firings before test), (3)new standard (uncoated) graphite tubes, and (4) new pyrolytic graphite-coated graphite tubes wlth standard pyrolytic graphite platforms. Samples analyzed as (A) aqueous standard (150 pg of Cr), (B) whole blood with a 2-fold dilution (94 pg of Cr), (C) urine with a 10-fold dilution (85 pg of Cr), and (D)mineralized bone with a 10-fold diiutlon (140 pg of Cr). Fast furnace temperature program employed (Table 1). Neither anaiyte isoformation nor background correction used.
whole blood Cr (9.4 pg/L, 94 pg), urine Cr (8.5 pg/L, 85 pg), and Cr in mineralized bone (1 4 pg/L, 140 pg) were obtained using the DABC design, the fast furnace temperature program (Table l), and neither analyte isoformation nor background correction (Figure 2). The best absorbance-time profiles for Cr, with respect to the shape of the atomization pulse (fairly symmetric with minimum tailing), and similar tapp (1.15 s, Figure 2) were obtained for Cr when atomization was performed in new and used pyrolytic graphite-coated graphite tubes (wall atomization); however, sensitivity achieved with
the used coated tubes was lower (- 18%) than that of the new tubes, probably because Cr interaction with carbon from the graphite tube wall, stripped of its pyrolytic graphite coating, was stronger. In this sense, it is thought that Cr-C interaction could lead to an incompletevolatilization of the analyte element due to the formation of thermally stable Cr compounds (e.g., Cr& etc.). The faster Cr atomization alsooccurred incoated graphite tubes, being slower in wall atomization from uncoated graphite tubes (1.25 s, Figure 2) and much slower in platform atomization (2.38 s, Figure 2). In Figure 2, profiles for Cr atomized from uncoated graphite tubes appeared unsymmetric and Cr atomization was delayed, presumably all due to the stronger Cr-C interaction taking place in this type of graphite tube. Figure 2 also shows that chromium atomization from a standard platform produced distorted and much more delayed absorption peaks (with respect to wall atomization) with pronounced tailing, plausibly due to the insufficient temperature available to form analyte atoms rapidly. In the ZEBC design, profiles for Cr resembled those obtained in the DABC design for platform atomization. In general, wall atomization in coated graphite tubes must be preferred for an adequate Cr atom formation. Carbon disfavored Cr atomization in ETA-AAS, in extreme opposition to the behavior exhibited by Pb, as described recently by Granadillo and Romero.8 Thus, one could infer that Cr atomization in the graphite furnace did not proceed via carbon-induced reduction of the atomic precursor to Cr atom. Coated graphite tubes tended to have a lifetime of >600 firings because carbon from the graphite surface was not used up by Cr during the graphite furnace analysis. Further research should be carried out to elucidate a possible atomization mechanism for Cr atomization in the graphite furnace; this was not the intention of the present paper. Furthermore, we presume that tubes made of materials different from graphite (e.g., tungsten) are able to be used for Cr determination by ETA-AAS. We assume that this nonorthodox approach could beapplied to other refractory metals that do not atomize via carbon Analytical Chemistry, Vol. 66,No. 21, November 1, 1994
3629
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1
Table 4. Accuracy of the Determlnatlon of Cr by Fast Furnace Program ETA-AAS Using the DABC and ZEBC Background Correction Instrumental Deslgns.
Cr concnb (pg/L) ref materialC
x
.-*
60
3
d
certified value
SRM 909
1.08 f 0.04
SRM 2670
8.5
OSSD 20/21
1.02 f 0.1 1
NIES No. 2
3.0
NIES No. 7
0.60"
NIES No. 8
7.8 f 0.5
NIES No. 9
0.80d
* 0.6
40
8 i .-
5
-
20
r L
0
m + "
0
40
20
60
80
100
Total chromium concentration by DABC design (pa) Figure 3. Comparison of results obtained for total Cr In 0, whole blood (23 samples); 0 , red blood cells (5 samples); W, urine (23 samples); and , mineralized bone specimens (7 samples), using fast furnace program ETA-AAS in the ZEBC (yjand DABC (x)instrumentaldesigns with neither analyte isoformationnor backgroundcorrection. Correlation 0.0839; r = 0.9926; n = 58; and p < as follows: y = 0.9667 x 0.0001. The standard error of the estimate (SJ is 0.2380, and the standard deviations of intercept and slope are 0.0494 pg/L Cr and 0.0158, respectively.
+
interaction with the atomic precursor, attempting in these cases only against the stabilized temperature platform furnace (STPF) concept23in three of its basic paradigms: isoformation, background correction, and platform atomization. Evaluation of the Analytical Parameters. Real samples were analyzed by using the DABC and ZEBC instrumental designs, fast furnace programs, and neither analyte isoformation nor background correction. No significant differences (p > 0,0001) were observed between total Cr concentration data (pg/L) obtained with the two analytical procedures followed (Figure 3). Accuracy was verified by analyzing seven standard reference materials. Results are shown in Table 4 and attest to the excellence of the analytical methods. The reliability of the analyses was further assessed through recovery studies. This was done by performing triplicatedeterminations of Cr in different Cr-spiked aliquots of two real whole blood and urine samples, all of them analyzed by using both instrumental designs. The average recoveries were 100% (range 94-103%). The within- and between-run (day-to-day) precisionvalues for the determination of Cr by fast furnace program ETAAAS in real samples of whole blood and urine and in three standard reference materials are shown in Table 5. Analyses were done in both instrumental designs and neither analyte isoformation nor background correction was employed. For the DABC design, the average RSD was 3.1% (corresponding to -0.02 pg/L Cr) while the average RSD for the ZEBC design was 3.7% (corresponding to -0.03 pg/L Cr), for both the within- and between-run precision, and for Cr concentrations ranging between 0.15 and 8.5 pg/L. The results can be considered adequate for this kind of analysis. Calibration graphs prepared with aqueous Cr standards were found to be linear up to 20 pg/L Cr (for the DABC (23) Slavin, W.; Manning, D. C.; Carnrick, G. R. At. Specfroxc. 1981, 2, 137.
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Analytical Chemistry, Vol, 66, No. 21, November 1, 1994
* 0.2
measured value DABC ZEBC 1.05 f 0.02 (n = 8) 8.4 f 0.4 (n = 7) 1.01 f 0.05 (n = 10) 3.2 f 0.1 (n = 8 ) 0.58 0.07 (n = 9) 7.9 0.8 (n = 7) 0.83 f 0.06 (n = 8)
* *
1.11 f 0.05 (n = 5 ) 8.9 0.2 (n = 9) 0.98 f 0.01 (n = 4) 3.1 0.3 (n = 7) 0.61 f 0.05 (n = 4) 7.8 f 0.3 (n = 5 ) 0.87 0.09 (n = 5)
* *
*
Neither analyte isoformation nor background correction performed. No significant differences between instrumental designs observed (g> 0.01). n, number of test portions analyzed; seven analyses of each portion were made. In diluted samples of SRM 909 Human Serum, SRM 2670 Toxic Metals in Freeze-Dried Urine, OSSD 20/21 Control Blood, NIES No. 2 Pond Sediment, NIES No. 7 Tea Leaves, NIES No. 8 Vehicle Exhaust Particulates, and NIES No. 9 Sargasso. Provisional value.
design) and up to 50 pg/L Cr (for the ZEBC design). All test portions analyzed were within these linear ranges. For the DABC design, the integrated absorbance, QA (s), was found to be linearly related to the concentration, C (pg/L Cr), by the equation QA = 0.0155C + 0.0002 (the standard deviations of intercept and slope were 0.000 01 and 0.000 16, respectively; r = 0.9998, p < 0.001); for the ZEBC design, QA = 0.01 19C 0.0003 (the standard deviations of intercept and slope were 0.0014 and 0.000 10, respectively; r = 0.9999, p < 0.001). The study of nonspectral interferences was done by comparing the slopes of the calibration graphs with those obtained by the method of standard additions. All biological and clinical materials under study (three aliquots each) and the standard reference materials were analyzed by the method of standard additions by use of both instrumental designs. The slopes of the standard addition graphs were identical to those of the aqueous standard calibration graphs. This implied the absence of nonspectral interference in the ETA-AAS analyses in these types of samples under the proposed analytical conditions and permitted the use of either calibration graphs or the standard additions method for quantification. For the DABC design, the limit of detection, defined as three times the standard deviation of a blank solution, was 0.03 pg/L Cr for a 10-pL injection volume of test solution. The characteristic mass was calculated to be 2.7 pg of Cr, which is in good agreement with the value reported of 3.5 pg of Cr.I4 For the ZEBC design, the limit of detection and the characteristicmass were0.2 pg/L and 5 pg, respectively.Slavin and Carnrick" reported a characteristic mass of 3.3 pg, using Zeeman background correction, Mg(NO& as analytical isoformer, and platform atomization. Using Zeeman correction, Pd-induced Cr isoformation and platform atomization, Berglund et al.24reported a mean characteristic mass for Cr of 4.3 pg.
+
(24) Berglund, M.; Frech, W.; Baxter, D. C. Spectrochim. Acto 1991,468, 176717.
Table 5. Wlthln- and Between-Run (Day-to-Day) Preclrlon Values for the Fast Furnace Program ETA-AAS Detennlnatlon of Cr In Sample8 and Reference Materlals Udng DABC and ZEBC Instrumental Deslgnra within-rune between-runsd
mean Cr concnb ( p g / L )
S D (re/L)
RSD (%)
SD ( P g m
RSD (%)
Real SamplesC Whole Blood 1 2 3
1.43 (1.40) 1.86 (1.90) 2.47 (2.55)
0.03 (0.05) 0.08 (0.06) 0.09 (0.09)
2.1 (2.1) 4.3 (3.2) 3.6 (3.5)
0.05 (0.06) 0.08 (0.09) 0.1 1 (0.10)
3.5 (3.6) 4.3 (4.7) 4.5 (3.9)
0.75 (0.80) 1.45 1.52 (1.55)
0.02 (0.06) 0.03 0.03 (0.03)
2.7 (7.5) 2.1 2.0 (1.9)
0.04 (0.07) 0.05 0.06 (0.04)
5.3 (8.8) 3.5 4.0 (2.6)
0.5 (1.9) 0.6 (0.5) 3.1 (3.8)
0.07 (0.08) 0.06 (0.12) 0.06 (0.09)
6.7 (7.4) 0.7 (1.4) 1.9 (2.9)
Urine 1 2 3
Reference Materials blood OSSD 20/21 urine SRM 2670 pond sediment NIES No. 2
1.05 (1.08) 8.50 (8.40) 3.20 (3.12)f
0.01 (0.02) 0.05 (0.04) 0.10 (0.12)
Values in parentheses are for ZEBC. Neither analyte isoformation nor background correction performed. b In diluted samples. Pentaplicate samples; three runs each. Triplicate samples per analysis; five runs each and analyzed over a five-day period. e Randomly collected from three volunteers. f In mineralized sample.
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By using fast furnace program ETA-AAS as proposed, 120samples could be analyzed in an 8-h working day. Faster furnace program ETA-AAS allowed us to analyze >200 samples/da y. Analytical Results. The proposed methods were used in the laboratory to establish the Cr ranges (pg/L Cr) of 20 chronic renal failure patients (CRF) from the Dialysis Unit at the Maracaibo University Hospital and 20 diabetic subjects (D) and 20 healthy adults (C) of Maracaibo City in whole blood (CRF 1.8-15.7; D 3.5-17.8; C 4.0-5.1), blood plasma [CRF 1.2-7.9 (50% of samples were undetectable); D 0.64.5; C 0 . 7 4 3 1 , blood serum [CRF 0.6-3.1 (39% of samples were undetectable]; D 1.8-13.4; C 0.5-2.01, red blood cells (CRF 2.1-20.9; D 3.0-7.0; C 3.0-7.0), bone (CRF 1.7-1 1.0 pg/g; D no biopsies performed; C 0.2-2.9 pg/g in autopsy samples), and urine (CRF no urine output; D 12.0-32.6; C 2.0-9.8). Chromium concentrations (mean f SD, pg/L) of one diabetic CRF patient were also considered: whole blood (4.2 f OS), blood plasma (undetectable), blood serum (13.3 f 0.8), and red blood cells (10.1 f 0.5). Chromium concentrations in the clinical materials analyzed from Maracaibo residents were higher than Cr data reported for other countries (e.g., urine Cr in the U S . population
