Determination of chromium in biological materials by atomic

for the quantitative determination of chromium. The absolute sensitivity of the method is 44 picograms chromium with a detec- tion limit of about 2 pi...
0 downloads 0 Views 722KB Size
Download PDF
Determination of Chromium in Biological Materials by Atomic Absorption Spectrometry Using a Graphite Furnace Atomizer I. W. F. Davidson and W. L. Secrest Department of Pharmacology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, N.C. 27103

A microanalytical method for the measurement of chromium in blood plasma, urine, and other biological materials.utilizing a flameless atomic absorption technique has been developed. The method combines the inherent specificity and simplicity of atomic absorption analysis with the greatly increased sensitivity possible with the heated graphite tube atomizer to provide a simple, rapid means for the quantitative determination of chromium. The absolute sensitivity of the method is 44 picograms chromium with a detection limit of about 2 picograms chromium. For routine analysis of plasma, the method is effective with either pre-ashed samples or direct sample analysis of very small amounts on the order of 20 to 200 PI. The relative standard deviation for quantities of chromium ranging from 285 to 690 picograms in 50 PI of wet ashed plasma is 5.2%. The method has also been used tor the determination of chromium in urine, whole blood, and other tissue samples. THEOCCURRENCE AND ROLE of chromium in normal body metabolism has recently been extensively reviewed by Mertz ( I ) . This element is considered essential for maintenance of normal physiological functions, and a nutritional deficiency has been implicated in a number of disease states (2-6). Evaluation of the importance of chromium in human nutrition has been hampered by the difficulties of determining the element in plasma, urine, and other biological materials where it exists a t concentrations of only a few parts per billion. A need exists for a method to determine chromium more rapidly and with greater ease than current techniques allow, and with sufficient sensitivity to be applied t o very small biological samples on a routine basis. The most widely used techniques for chromium determinations in biological materials, atomic absorption (7) and emission spectrometry (8),have recently been refined and improved in sensitivity (9, 10). New methods of greater sensitivity have been proposed based on gasliquid chromatography of chromium chelates (11-15). How(1) W. Mertz, Physiol. Reo., 49, 163 (1969). (2) L. L. Hopkins, 0. Ransome-Kuti, and A. S. Majaj, Amer. J. Clin. Nutr., 21, 203 (1968). (3) W. H. Glinsmann and W. Mertz, Metab. Clin. Exp., 15, 510 (1966). (4) R. A. Levine, D. H. P. Streeten, and R. J. Dorsey, ibid., 17, 114 (1968). ( 5 ) G. D. Martin, J. A. Stanley, and I. W. F. Davidson, Invest. Ophthal., 11, 153 (1972). (6) I. W. F. Davidson, M. Lang, and W. L. Blackwell, Diabetes, 16, 395 (1967). (7) F. J. Feldman, E. C. Knoblock, and W. C. Purdy, Anal. Chim. Acta, 38, 489 (1 967). (8) I. H. Tipton, M. J. Cook, R. L. Steiner, C. A. Boye, H. R. Perry, and H. A. Schroeder, Health Phys., 9, 89 (1963). (9) F. J. Feldman, Presented at the VI1 International Congress of Nutrition, Prague, 1969. (10) K. M. Hambidge, ANAL.CHEM., 43, 103 (1971). (11) J. Savory, P. Mushak, and F. W. Sunderman, J. Chromatogr. Sci., 7, 674 (1969). (12) J. Savory, P. Mushak, F. W. Sunderman, R. H. Estes, and N. 0. Roszel, ANAL.CHEM.,42, 294 (1970). 1808

ever, a number of factors have restricted widespread, routine use of these methods. Atomic absorption spectrometry requires pre-concentration of the sample by ashing, oxidation, chelation, and extraction into a n organic solvent, all of which prolongs analysis time and increases risks of contamination. Although of greater potential sensitivity, gas chromatographic techniques also demand extensive sample preparation, and for routine use the current analytical techniques are unduly complex and time consuming. A critical evaluation of gas chromatographic procedures for chromium has been made recently by Savory et al. (16). The difficulties associated with the analysis of chromium in biological materials by flame atomic absorption spectrometry occur because the amounts of chromium in biological samples are close t o the detection limits of present instrumentation with flame atomization. The recent introduction, of flameless atomizing devices (17-22) that produce a population of ground state atoms more efficiently than the nebulizer-flame sampling methods have greatly improved the sensitivity of atomic absorption measurements. A several hundredfold improvement in absolute sensitivity for chromium has been reported with an electrically heated graphite tube atomizer (17, 18). With this atomizer, the sample is introduced directly into the tube, and provision is made for programming the temperature of the tube for the sequential operations of sample drying, ashing, and atomization. Beside the greater sensitivity afforded by the increased efficiency of atomization, other potential advantages of the graphite tube atomizer for the analysis of chromium in biological fluids lie in the small sample volume required (Le,, 5 to 50 pl) and the potential for analyzing samples containing organic matter without pre-treatment. The purpose of this study was to evaluate the graphite tube atomizer as a practical means of performing the quantitative determination of chromium in blood plasma and urine. Some difficulties encountered with the direct analysis of plasma and urine samples are described. A more suitable method was developed incorporating a simple wet ashing procedure that permitted rapid analysis, free of chemical and physical (13) G. H. Booth, Jr., and W. J. Darby, ANAL.CHEM., 43,831 (1971). (14) L. C. Hansen, W. G. Scribner, T. W. Gilbert, and R. E. Severs, ibid., p 349. (15) W. R. Wolf, M. L. Taylor, B. M. Hughes, T. 0. Tierman, and R. E. Severs, ibid., 44, 616 (1972). (16) J. Savory, M. T. Glenn, and J. A. Ahlstrom, J . Chromatogr. Sci., 10, 247 (1972). (17) F. J. Fernandez and D. C. Manning, At. Absorption Newslert., 10, 65 (1971). (18) Ibid., 9, 65 (1970). (19) B. Welz and E Wiedeking, Fresenius' 2. Anal. Chem., 252, 111 (1970). (20) H. L. Kahn, Amer. Lab., 3, 35 (1971). (21) M. D. Amos, P. A. Bennett, K. G. Brodie, P. W. Y . Lung, and J. P. MatouSek, ANAL.CHEM., 43, 211 (1971). (22) J. Y . Hwang, P. A. Ullucci, S. B. Smith, and A. L. Malenfant, ibid., p 1319. ~

ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972

interferences, and which was generally applicable to biological samples other than plasma and urine as well. PLASMA W I T H ADDED STANDARDS C,6+

EXPERIMENTAL Apparatus. The design features of the Perkin-Elmer H G A 70 graphite tube furnace have been described (17-20). This atomizer is essentially a modification of the heated graphite tube of Massmann (23, 24). The furnace was mounted on a P-E Model 290 B single beam atomic absorption spectrometer, with provision for an undampened signal output to be recorded with a Varian Model 825, IO-inch chart recorder with a pen response of 0.5 second. The signal output to the recorder was amplified 4-fold by a fixed scale expansion provision of the instrument. Additional signal amplification was obtained with the recorder. Determinations were ordinarily performed at an appropriate recorder expansion to provide a total signal amplification of 8-fold (0 to 62.5 milliabsorbance) or 20-fold (0 to 25.0 milliabsorbance). Tbe spectrometer was operated with a slit width of 20 A, a t the primary wavelength of chromium, 357.8 nm. The chromium hollow cathode lamp (P-E Intensitron) was operated at 15 milliamperes. During sample atomization, incandescence from the heated graphite tube of the furnace produced an emission which overlapped the wavelength for chromium absorption. A Corning filter C. S. 7-60 with a band pass width of 60 nm was placed in the filter holder at the entrance to the monochromator compartment. The filter reduced by 30% the intensity of the sample beam to the monochromator. The loss of beam intensity was compensated in part by a high lamp intensity and a wide slit width. Nitrogen was used to provide an inert gas atmosphere in the furnace and to purge the graphite tube at a flow rate of 1.5 l./min. Reagents. Standard stock solutions containing 10 mg of Crjml were prepared in 0.3N HC1 (Ultrex) from reagent grade chromium salts : Cr(VI), potassium dichromate; Cr(III), chromium potassium sulfate; and Cr(II), chromous chloride which was prepared immediately before use. Secondary working standard solutions were prepared by dilution of stock solutions with 0.3N HCl (Ultrex). The water used was double distilled and demineralized. All solutions and biological samples for analysis were stored in polyethylene. The concentrated acids used were of ultra-high purity; HCI (Ultrex, Baker Chemical Co.) and 70% HC104 (G. Frederick Smith Chemical Co.). Hydrogen peroxide (30 %) (Fisher Scientific Co.) with a low chromium content confirmed by analysis was used. Procedure. PREPARATION OF BIOLOGICAL SAMPLES.Blood samples were drawn from donors with 10-ml Vacutainers (Becton, Dickinson and Co.), and from experimental animals with 1-ml disposable syringes with aluminum needles. Heparin was used as the anticoagulant. Plasma samples were obtained by centrifuging freshly drawn heparinized whole blood. They were stored at 4 "C in plastic tubes when analysis could be performed within 8 hours or frozen and kept at -20 "C. No differences in Cr content were noted between frozen and fresh plasma. Urine was collected from donors in plastic bottles, acidified, and stored at 4 "C. Wet ashing was carried out in two-dram screw-top vials with Teflon-lined caps (17 x 60 mm, Scientific Specialties Inc.). The vials were acid washed to minimize chromium contamination and provide a consistently low blank. To the vials were added 200 p1 of plasma or urine followed by 400 p1 of perchloric acid. The reagent blanks were prepared similarly with 200 MI of distilled demineralized water. The vials were placed in an aluminum heating block (Fisher Scientific Co.) maintained at 135 "C. After 45 minutes, the

(23) H. Z. Massmann, F r e s e h s ' Z . Atial. Clzem., 225, 203 (1967). (24) H. Z. Massmann, Spectrochim. Acta, 23B, 215 (1968).

I-

Figure 1. Tracings of recorder response showing the reproducibility of graphite furnace atomization of plasma chromium and aqueous chromium standards. Independence of the response to different valency states of chromium and chromium recovery after addition to plasma is demonstrated Plasma, wet ashed sample, equivalent to 25 pl; chromium aqueous standards, 200 pg as Cr(1I)Cl2, Cu(LII)K(S04)2, or K2Cr(VI)OI; plasma peak height, 65 mm with 8-fold signal amplification

vials were removed, allowed to cool, and 100 pl of hydrogen peroxide was added to each vial. They were heated for an additional 15 minutes at 135 "C, and then the temperature was raised to 180 "C and the acid digest allowed to reflux for 45 minutes. The digest was evaporated to a dry ash by adjusting the temperature to 215 "C for approximately 30 minutes. After cooling to room temperature, 400 p l of 0.3N HC1 was added and the vials were capped. Solution of the dry ash was complete, and the chromium content of the solution was stable indefinitely. Plasma samples of 50 p1 were wet ashed as above in onedram vials (14 X 45 mm, Pierce Chemical Co.) with 100 pl of perchloric acid and 50 pl of hydrogen peroxide. SPECTROMETRIC DETERMINATIONS. Sample aliquots (50 p1 or less) were introduced into the furnace with Eppendorf micropipets. Temperature program seven was adopted for samples which did not contain organic matter. The program cycle was: sample drying, 2 seconds per p1 solution (at ca. 100 "C); dry ashing, 60 seconds (at ca. 1100 "C). Optimum atomization voltage for chromium was determined by experiment to be 12 seconds at 8.5 volts (about 2400 "C). However, except for standard solutions of chromium, the matrix of unknown samples (plasma, urine, or wet ashed samples) often produced interference peaks close to and sometimes superimposed on the chromium peaks. These peaks were presumably due to smoke formation through reactivating, during atomization, condensed or sublimed matrix material from the ashing phase. These interferences could not be eliminated by any modification of the dry ashing phase, but they were eliminated by adopting a double atomization procedure. After drying and ashing, the sample was atomized for 12 seconds at 4.5 volts (about 1400 "C); the cycle was then repeated as follows: drying 10 seconds; dry ashing, 30 seconds; and atomization 12 seconds a t 8.5 volts (2400 "C). The initial atomization at 1400 "C was not a sufficient temperature to volatilize chromium or chromium salts and loss of chromium was not encountered. Since the graphite tubes occasionally varied in electrical conductance, optimal atomization voltages must be reestablished for each tube using standard chromium solutions. The absorption peaks were measured from the bottom of the initial negative spike to the maximum height. While wet ashed samples were preferred for analysis, diluted plasma samples (50 pl or less) were analyzed by

ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972

1809

I

-Full

Scate(10in.)

60 I I

I

I

/

/

I

/

50i 50

20X scale expansion

-18

Table I. Composition of Salt Solution Used to Investigate Ion Interference Effects in the Graphite Atomizera Salt Concentration, ppm NaCl 6700 CaC03 12000 CaHP04.2H20 3000 CuSOa * 5Hz0 12 FeS04(NH4)2S04. 6H20 1100 MgSOa * 7Hz0 4076 MnS04 4H20 200 KI 32 KzHP04 12900 ZnCh 12 a 497, salt mixture in 0.3N HCl.

f', 8X scale expansion

/

20 20-1

RESULTS AND DISCUSSION 10

C

100

200

300

400

CHROMIUM, picograms

Figure 2. Calibration curves for determination of chromium with the graphite atomizer for two different sensitivity ranges

K2Cr(VI)04in 0.3NHC1,lO ng Cr/ml Cr(II)CI2in 0.3NHC1,lO ng Cr/ml Cr(III)K(S04)2in 0.3N HCI, 10 ng Cr/ml 0 K2Cr(VI)04added to 497, salt mixture (Table I) in 0.3N HCl, 10 ng Cr/ml 0 A 0

- F u l l s c a l e ( i 0 in.)

/

60 24

50

expansion

18 w

2 40 4

m (L

0 v)

._ --

3c

/

-

I

2c

10

(

Figure 3. Determination of chromium in wet ashed plasma, 0 , and urine, 0, showing the proportional response with increasing amounts of wet ashed sample introduced into the graphite atomizer

atomic absorption after direct introduction into the graphite atomizer. Human plasma was diluted 1 :1 with water and monkey plasma 1 :3. Program seven was modified as follows: sample drying, 4 seconds per p1 plasma sample; dry ashing, 6 minutes; atomization 30 seconds at 4.5 volts. The second atomization cycle was carried out as described for wet ashed samples. 1810

Initial experiments were concerned with establishing the characteristics of the graphite tube atomizer for chromium. The tracings shown in Figure 1 of actual atomic absorption peaks obtained from replicate samples of aqueous chromium standards and of a wet ashed plasma sample demonstrate the reproducibility of measurements with the graphite atomizer. A large undulating recorder response preceding the chromium peak, that is due to moisture and smoke from the sample drying and ashing, is not shown, as the recorder is customarily turned on 30 sec before the atomization. The precision of instrument response for replicate aqueous chromium standards, 100 to 200 pg, was 2.1 % and 1.2% re1 std dev, respectively, for sample volumes of less than 50 ~1 introduced into the atomizer. Because of the high instrument precision, the routine procedure adopted was to make 3 determinations in the furnace for each sample and to average the absorption peak heights. The height of the absorption peak was directly proportional to the absolute amount of chromium introduced into the atomizer, from the detection limit up to 400 picograms at 8fold signal amplification, and up to 200 picograms at 20-fold signal amplification (Figure 2). Figures 1 and 2 also show that the height of the absorption peak for chromium was independent of valency state for these ranges. Chromium may occur in different valency states in biological materials. For aqueous solutions of the element as a salt, the sensitivity for chromium was 44 X 10-'2 gram for a signal of 1 % absorption (4.4 milliabsorbance) (Figure 2). The practical detection limit was about 2 picograms with signal amplification of 20fold. For the practical application of the method to the measurement of chromium in biological materials, spectral and chemical interferences from the presence of extraneous ions in the matrix of these samples were studied briefly. Spectral interference may arise from absorption due to light scattering and molecular absorption, and it results in a nonspecific absorption peak by decreasing the vaporization rate of chromium or by incomplete dissociation of the chromium salts present during atomization, Shown in Figure 2 are the results of recovery experiments for chromium added to a concentrated salt solution containing the principal ions of biological samples. The absorption peak for chromium was not affected by the presence of 500- to 5000-fold excesses of cations Na, Ca, Cu,Zn, Fe, Mg, Mn, K, or by the anions C1, I, COS,HPO4, so4. The calibration curve for chromium in the presence of these ions was identical with the curve obtained with chromium standard solutions. The aggregate total ion concentration constituted a 4 % salt solution (Table I), which was a salt

ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972

Table 11. Determinations of Chromium in Human Blood Plasma and Urine Cr content Wet Recoverv added Cr ashed Total soh analyzed Sample, Added found Recovered ng/mla Pg Sample Pga pg*zb Plasma

z

S . B. W. D. J. T. B. W L. B.

L. s. I. D. Average

173,5 101 .o 126.0 103.3 77.5 126.5 179.8

6.94 4.04 5.04 4.13 3.10 5.06 7.19 5.07

200 200 200 200 200 200 200

371.3 289.7 324.6 301.3 278,3 328.9 384.6

98.9 99.4 99.3 99.0 100.4 101.2 102.4 100. 1 1 1 . 3 std dev

144.5 266.0 180.3 80.8 131.5 80.8 60.0 137.3 101.3 107.3 150.5 122.3

5.78 10.64 7.21 3.23 5.26 3.23 2.64 5.49 4.05 4.29 6.02 4.89 5.23

100 100 100 100 100 100 100

250.2 360.5 280.4 183.5 230,O 174.9 163,l 237.3 198.4 207.3 253,6 222.3

105.7 94.5 100. 1 103.7 98.5 94.1 97.1 100.0 97.1 100.0 103.1

Urine E. A. D. I. R. R. L. Y . B. S .

Figure 4. Analytical curves for direct determination of plasma chromium with the graphite atomizer by comparison with aqueous chromium standards and by the method of additions A . Chromium aqueous standards E . Human plasma, diluted 1:l with water C. Plasma, 10 MI,with added aqueous chromium standards

s. s.

M. W. T. W. J. P. T. P. D. W. I. D.

Average a

100

100 100 100 100

(25) W . Mertz, Agricultural Research Service, personal communication. 1971.

99.4 i3 . 4 std dev

Minus blank, 33.9 pg.

* 50 pl wet ashed solution analyzed; concentration severalfold greater than to be expected in blood, urine, or other biological samples. Further investigation of matrix interferences was performed with wet ashed plasma and urine samples. Figure 3 shows that the absorption peak was directly proportional to the amount of wet ashed plasma or urine sample analyzed. When different amounts of chromium were added to a series of plasma and urine samples, total recovery was obtained (Table 11). Figure 1 shows that when chromium of different valency states was added to wet ashed plasma, recovery was also complete. The absence of significant spectral or chemical interference from sample composition demonstrated the effectiveness of the double atomization procedure, and confirmed that the method allows the determination of chromium in wet ashed samples of plasma and urine to be performed simply by comparison with aqueous chromium standards. The results of the application of the method to the quantitative determination of chromium in plasma and urine from normal human subjects are given in Table 11. Chromium concentrations in fasting plasma from 7 subjects ranged from 3.1 to 7.2 ng/ml with a mean of 5.1 ng/ml. These concentrations are similar to recent values obtained by other investigators by emission spectrometry and by the nebulizer-flame method of atomic absorption (10, 25). The chromium concentration in urine from 12 fasting normal subjects ranged from 2.6 to 10.6 ng/ml. Assuming an average 24-hour urine void volume of 1500 ml, the total chromium excretion approximated 4 to 16 pg/24 hours and these values are also similar to those obtained by other methods (10). The precision of the method as determined with replicate samples of normal plasma was 3.8 % re1 std dev (Table 111).

100.0

equivalent to 25

pl

plasma

or urine.

The pre-treatment of plasma and urine samples by the wet ash procedure described was both simple and effective, and without sample chromium loss. The data given in Table I1 show that the recovery of chromium added to plasma and urine samples before ashing was 100.1% and 99.4%, respectively, with re1 std dev of 1.3% and 3.4%. Pre-treatment of the sample by wet ashing, however, necessitated a blank correction that amounted to about 20% of the normal chromium content of plasma and urine samples (Table 111). Of the chromium found in the blank, only 50% could be attributed to the ashing reagents. Since the blank determinations were highly reproducible, it was concluded that acid leaching of chromium from the glass of the digestion vials contributed significantly to the blank. This was verified by coating the vials with silicone (Siliclad, Clay-Adams, Inc.). The blank was reduced to 0.41 i 0.12 std dev, ng/ml chromium, a value only slightly more than that given by the ashing reagents, 0.31 ng/ml chromium. Silicone coating was an effective means of minimizing the blank. For the determination of chromium in the plasma of small experimental animals, sample size often becomes restrictive. This is also the case when large survey studies are undertaken and samples obtained by finger puncture. The method described offers sufficient sensitivity for the determination of chromium in 50 ~1 of plasma with a precision of analysis of 5.2% re1 std dev (Table 111). Recovery of added internal standard to 5O-pl samples before wet ashing was 100.9% with a re1 std dev of 3.3%. This analytical reliability for 50-p1 samples compared favorably with that obtained with the larger pla:.ma sample volume, 200 p l (Table 111).

ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972

1811

Table 111. Precision of Replicate Chromium Determinations Cr found Vol. ashed, Total (-blank) soln analyzed, p1 Aliquot pg, mean pg, mean

No. of Plasma Cr, ng/ml, mean Sample samples Std solution Cr 200 Pg 20 100 Pg 20 Plasma (W. S.) 200 pla 20 50 178.5 1156.8 5.78 blank 20 50 33.9 50 plb 20 20 40.1 284.0 5.71 50 p1 added Cr, 400 pg 10 20 80.5 687.6 Recovery blank 20 20 11.7 100.9% a Each sample and blank was wet ashed, dried, and dissolved in 400 p1 0.3N HC1; scale expansion %fold. Each sample and blank was wet ashed, dried, and dissolved in 200 p1 0.3N HCI; scale expansion 20-fold.

+

Table IV. Determinations of Chromium in Squirrel Monkey Plasma No. of Cr content, Method animals ng/ml Range, ng/ml Wet ashed Chow fed 12 6.44 =t1.69 3.43-8.33 std dev Chow fed, Cr supplemented" 9 16.32 i 3.41 11.80-21.05 std dev Direct analysisb Chow fed 12 5.24 i 1.84 2.75-9.57 std dev Cr supplement in drinking water, 10 ppm. * Plasma diluted 1: 3 with water and analyzed without pretreatment by method of additions.

To demonstrate the capability of the method to measure differences in plasma levels of chromium, and also the utility of the method for animal experimentation, plasma chromium concentrations were determined for two groups of squirrel monkeys maintained under different experimental conditions. The monkeys were fed ordinary monkey rations (Purina Chow 25) for a 12-week period, but one group was provided also with a dietary chromium supplement in the form of 10 ppm chromium added to their drinking water. Blood samples, 0.3 ml, were drawn by venipuncture and the plasma aliquots for analysis, 50 p1, obtained by centrifugation. The results of the determinations of plasma chromium are presented in Table IV. For normal monkeys maintained on ordinary rations, plasma chromium concentrations were similar to the range found for humans, but the plasma levels for animals whose diet was supplemented with chromium were increased approximately 2.5-foId. Inherent in the design of the graphite tube atomizer is the potential for direct analysis of small plasma or urine samples (5 to 50 pl) without sample pre-treatment by ashing or other complex extraction and chelation procedures. In addition to these obvious advantages, direct sample analysis eliminates the necessity of reagent and other blank corrections. Figure 4 shows results of the analysis of human plasma chromium after direct introduction of the plasma untreated, except for dilution, into the atomizer. The absorption signal for chromium was observed to be directly proportional to increasing amounts of plasma. Addition of chromium standards to the plasma produced a curve parallel to a calibration curve for chromium. These results showed that a direct determination of chromium 1812

Re1 std dev, 1.2 2.1 3.8 22.1 5.2 i 3.3

20.0

in plasma could be performed simply by comparison with aqueous chromium standards or by the standard method of additions. For the plasma analyzed by these two methods (Figure 4), the same chromium concentration, 4.6 ng/ml, was obtained. However, the occasional human plasma sample exhibited matrix interferences with marked suppression of the absorption signal. This suppression could not be eliminated by greater plasma dilution or by any modification of the ashing cycle of the atomizer. The occurrence of matrix interferences was especially common with monkey plasma. But plasma exhibiting sample composition interferences could be analyzed by the standard method of additions, and this method provided values for plasma chromium concentrations in close agreement to those obtained after pre-treatment of samples by wet ashing. Table IV shows the analysis by the two methods of the plasma chromium of two groups of 12 normal fasting monkeys. The mean value for plasma chromium of the group analyzed by the method of additions did not differ statistically from that of the group determined by prior wet ashing. There are several disadvantages to the direct analysis of samples in the atomizer. Plasma samples cannot be determined reliably except by the standard method of additions, and the severe matrix interferences experienced with urine samples precluded the use of even this method. Moreover, the minimum sample volume of plasma required for the method of additions approximated 100 pl for a single complete determination, twice the sample size needed for wet ashed samples. Because of the far longer ashing cycle in the atomizer required for untreated plasma (6 min), the instrument analysis time for a single plasma sample analyzed by the method of additions averaged 35 min, us. 3 min for a wet ashed sample. The wet ashing procedure did not add greatly to total analysis time, since 40 samples may be ashed conveniently in a single batch. The direct analysis of plasma chromium was also associated with a greater precision error, due in part to a larger pipetting error, to uneven drying and splattering of the plasma sample in the graphite tube, and also to entrapment of chromium in the excessive residue remaining in the tube after several sample ashings. The useful life of a graphite tube when used for direct analysis was only 40 to 50 samples with frequent removal of residue buildup. Residue buildup did not occur in the graphite tube with wet ashed samples, and the useful tube life averaged 150 to 200 samples before deterioration could be detected with standard solutions of chromium. For these reasons pre-treatment of biological samples by the

ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972

simple and rapid wet ashing procedure described was preferred for the atomic absorption analysis of chromium with the graphite atomizer. This procedure had the further advantage that it was generally applicable to the determination of chromium in biological materials other than plasma and urine. It has been used as described with equal effectiveness to the determination of chromium in whole blood, red blood cells, and to 50-mg samples of other tissues such as human hair and finger nails. It is concluded that atomic absorption spectrometry with the graphite tube atomizer provides the basis for a simple and very rapid method for the quantitative measurement of chromium in biological samples. The inherent specificity and sensitivity of this technique satisfies the requirements for the determination of chromium in very small biological

samples at concentrations of a few parts per billion. Pretreatment of the sample by wet ashing is recommended because : a) chemical interferences due to sample composition are avoided, b) there is general applicability to a variety of biological materials, and c) considerable reduction of instrument analysis time with wet ashed samples occurs. For simplicity, sensitivity, reliability, and speed of analysis, the method as proposed has distinct advantages over other current procedures which require extensive sample preparation and analytical time.

RECEIVED for review March 27, 1972. Accepted May 25, 1972. This work was supported by National Institutes of Health Grant No. AM13322.

Ultrasonic Nebulization in a Low-Emission Flame for Atomic Fluorescence Spectrometry M. B. Denton’ and H. V. Malmstadt Deparfmenf of Chemistry, School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, Ill. 61801 The use of an ultrasonic nebulizer in conjunction with a low turbulence, argon-hydrogen-entrained air flame provides improved performance of almost two orders of magnitude as compared with a conventional pneumatic total consumption burner. Increased sensitivity results from more efficient nebulization, reduced light scattering, and lower flame background emission. An automated sample changing system is described which provides the required freedom from sample cross-contamination. THEHIGH SENSITIVITY in trace analysis offered by atomic fluorescence spectrometry AF ( I ) , has been demonstrated by many workers (2-4). Various different flame-burner-nebulizer combinations have been investigated for use in generating the required atomic vapor (3). Desirable atomizer characteristics include efficient conversion of sample matrix into analyte atoms suitable for excitation, low radiational background, a low concentration of quenchers, a long residence time of analyte atoms in the optical path, as well as low cost and simplicity of operation ( 4 ) . An ideal atomizer should also produce a minimum of scattering centers. This is particularly important when using high intensity sources in resonance line fluorescence (5) where any remaining droplets or salt crystals can scatter the exciting radiation into the entrance optics of the detection system.

Present address, Department of Chemistry, University of Arizona, Tucson, Ariz. 85721. (1) J. D. Winefordner and T. J. Vickers, ANAL.CHEM.,36, 161 (1964). (2) Richard Smith, “Spectrochemical Methods of Analysis,” J. D. Winefordner, Ed., Wiley-Interscience, New York, N.Y., 1971, Chapter 4. (3) J. D. Winefordner and T, J. Vickers, ANAL.CHEW,42, 206 R (1 970). (4) J. D. Winefordner and R. C. Elser, ibid.,43 (4),24 A (1971). (5) M. B. Denton and H. V . Malmstadt, Appl. Phys. Lett., 18, 485 (1971).

This requirement would tend to indicate the desirability of an extremely hot flame or plasma to ensure complete desolvation and conversion into atomic vapor; however, this increased temperature is often accompanied by an undesirable increase in background emission. For many applications, better results can be achieved in a low-temperature flame through generating an aerosol composed of very fine droplets of sample solution which can be rapidly and efficiently desolvated. The salt crystals resulting from desolvation of smaller droplets will, consequently, be smaller and, therefore, more readily converted to atomic vapor. Bratzel, Dagnall, and Winefordner ( 6 ) compared premixed and turbulent air-hydrogen and argon-air-hydrogen flames. They noted that turbulent flames suffered from several problems: having less than 100% atomization efficiency (particularly in the lower regions); being difficult to illuminate and collect light from the larger high regions ; and having very high rise velocities, shortening the time allowed for solvent and solute evaporation. However, the better results they achieved with turbulent flames were attributed to the higher total volume of solution reaching the flame. Considerable effort has been expended by many workers to characterize various types of nebulizers. Dean and Carnes (7) studied the drop-size distribution of the Beckman integral aspirator, total consumption burner and found that with water, 31 of the drops were over 20 pm in diameter. Stupar and Dawson (8) have contrasted pneumatic and ultrasonic methods for the production of aerosols. They have observed that ultrasonic devices generate more homogeneous aerosols. At higher ultrasonic frequencies, the resulting droplet size decreases (8, 9). The proper ultrasonic nebulizer offers a solution to the problem of producing very small droplets with (6) M. P. Bratzel, Jr., R. M. Dagnall, and J. D. Winefordner, ANAL. CHEM., 41.713 (1969). (7) John A. Dean and William J. Carnes, ibid.,34,192 (1962). (8) J. Stupar and J. B. Dawson, Appl. Opt., 7, 1351 (1968). (9) J. Spitz and G.Uny, ibid.,p 1345.

ANALYTICAL CHEMISTRY, VOL. 44, NO. 1 1 , SEPTEMBER 1972

1813