Direct Flameless Atomic Absorption Determination of Lead in Blood Jae Y . Hwang, Paul A. Ullucci, and Charles J. Mokeler Instrumentation Laboratory Inc., 113 Hartwell Avenue, Lexington, Mass. 021 73
In the past few years, the problem of lead intoxication has received considerable attention in this country and the effect of lead on human beings is well documented (1-3). Lead interferes with a number of body functions, most notably the central nervous system, the hematopoietic system and the kidney (2). Although there are conflicting theories on the choice of the biological samples, the determination of lead levels in whole blood is one of the most useful diagnostic tests (2-6). Various analytical techniques (7-25) have been applied t o this analysis as shown in Table I. The analytical method receiving considerable attention of late has been Atomic Absorption Spectrometry (AAS). The extreme specificity of AAS coupled with its good sensitivity makes it the method of choice. In spite of its good sensitivity, however, a separation or preconcentration step has been required in most cases for the determination of lead in blood. Table I reviews many of the new and existing AA techniques for this determination by giving the amount of blood necessary for the analysis as well as a brief description of each procedure involved. The most promising techniques with special regard to time and amount of sample required are the semi-flameless or flameless procedures (19-25). 'Two direct procedures using flameless AA and involving sample dilution only have been developed. Both procedures make use of the carbon rod atomizer; one involves atomic fluorescence (22) and the other atomic absorption (24). To determine lead in blood with the last two techniques, two separate absorbance readings are made: one of the lead plus Goyer, Amer. J. Pathoi.. 64, 167 (1971). P. P. Craig and E. Berlin, Environment, 13, 2 (1971). J. J. Chisoim. Jr., Sci. Amer., 224, 15 (1971). J. B. Hursh, A. Schraub, E. L. Sattler, and H . P. Hofmann, Heaith Phys.. 16, 257 (1969). A . A. Moncrieff. P. 0. Koumedis, B. E. Clayton, H . D. Patrick, A . G . C . Renwick, and G . E. Roberts, Arch. Dis. Childhood, 39, 1 (1964). Committee Report of the American Academy of Pediatrics, 44, 291 ( 1 969) J . C . Wells and R. E. Seidner, Develop. Appl. Spectrosc., 5 , 333 (1965). M . Roschig and H . Matshiner, Z. Med. Labortech., 11, 7 (1970) K. Horiuchi, S. Horiguchi, K. Shinagawa, F. Takada, and K . Teramoto. Osaka City Med. J . , 14, 113 (1968); (Chem. Abstr., 71, 6549 (1 969)). R. G . Keenan, D . H . Byers, B. E. Sattzman, and F. L. Hyslop, Amer. lnd. Hyg. Ass. J.. 24, 481 (1963) E. B. Sandeii, "Colorimetric Determination of Traces of Metals." 3rd ed., Interscience, New York, N . Y . 1959. E. Berman, Amer. J. Ciin. Pathol., 36, 549 (1961). E. Berman, A t . Absorption Newslett., 3, 111 (1964) E. Berman. V . Valavanis, and A . D u b i n . Ciin. Chem., 14, 239 (1968). R. 0 . Farreliy and J. Pybus, Ciin. Chem., 15, 566 (1969). D. W. Hessel, A t . Absorption Newslett.. 7, 55 (1968). J . J. Chilsom. unpublished works. H . Lyons and F . E . Quinn, Clin. Chem., 17, 152 (1971). S. Hilderbrand, S. R . Koirtyohann. and E. E. Pickett, Biochem. Med., 3, 437 (1970). J . Y . Hwang, P. A. Ullucci, and A . L. Malenfant, Instrumentation Laboratory Reprint, "Atomic Absorption Spectrometric Determination of Lead in Blood by theSolid Phase Sampler Technique" (1970). H. T . Delves, Analyst, (London). 95, 431 (1970). M. D. Amos, P. A. Bennett, K. G . Brodie. P. W . Y. Lung, and J . P . Matousek, Anal. Chem.. 43, 211 (1971). J. Y . Hwang, P. A . Ullucci, S. B. Smith, Jr., and A. L. Malenfant, Anal. Chem., 43, 1319 ( 1 9 7 1 ) . N. P. Kubasik, M . T . Volosin, and M . H . Murray, Ciin. Chem., 18, 410 (1972). E. Norvai and L. R. P. Butler, Anal. Chim. Acta.. 58, 47 (1972). R. A .
background absorption and the other of the background absorption only. These signals are later subtracted out. The result is attributed to the lead only. In one of these techniques (24), it is recommended that a working curve be established for a set of unknown samples. With this in mind, the present paper is purported to present a method which eliminates the needs of double measurements to determine lead in blood and of establishing a working curve for each set of unknowns. To eliminate both needs, an automatic background corrector and frequent standardization techniques are employed in this study. The accuracy of the present technique was checked by the results obtained by other analytical techniques using actual blood lead standards that are available.
EXPERIMENTAL All AA measurements were made with an Instrumentation Laboratory (I. L.) Model 353 dual-double beam AA Spectrophotometer, The hurner head of the instrument is replaced with a n I. L. Model 355 Atomization Chamber. Both systems have been previously described in the literature (26, 27). T h e analytical parameters are given in Table 11. The background correction mode is used as described in the I. I,. Instruction Manual. The means by which the AA Spectrophotometer automatically corrects the signal is shown in Figure 1. In the analytical channel, using a lead hollow cathode lamp, the signal is affected by both lead and background absorptions. In the reference channel, using a hydrogen continuum lamp, the only effect is from the background absorption. Since both light sources are pulsed a t different frequencies, they can be electronically separated and the background absorption automatically subtracted. Alignment of the Atomization Chamber. Alignment of the chamber is critical for the maximum sensitivity. The chamber is visually aligned with the optical path. A narrow cardboard strip is placed and allowed to rest on the tantalum ribbon. The chamber is then adjusted so that the light beam from the hollow cathode tube is right above the center of the tantalum ribbon. Surface contamination of lead on the ribbons can be eliminated prior to absorption measurements by heating the ribbons in argon gas a few times around 2000 "C. Procedure. Sample Running. A 25-fil sample of 1-10 water diluted whole blood is placed on the tantalum ribbon by means of a micropipet. A 1-10 dilution of whole blood is used not only to facilitate the manipulation of viscous blood samples but also to complete hemolysis of red blood cells (28). The proper drying and ashing temperature, about 400 "C (DRY scale 5-7 on power supply). is predetermined by spiking a blood sample with lead standard. The optimum drying temperature is the highest temperature a t which no lead is lost. Atomization temperature is set a t about 1500 "C. When all other parameters are set, the ANALYZE button is pressed and the signal appears in one minute. After several analyses, a small carbon residue will appear which is simply brushed off. Heating the ribbon in hydrogen gas a t a maximum temperature of about 2000 "C before use and periodically during its use will increase the ribbon life to more than 200 analyses. Standardization. The method of standard additions w'as used to establish lead concentrations. One sample is analyzed by this method which serves a s a standard for the rest of the samples. Blood samples of known lead concentrations were used as standards to check the validity of the above standardization technique.
(26) S. B. Smith, Jr.. J. A . Blasi, and F. J. Feldman, Anal. Chem., 1525 (1968). (27) J. Y . Hwang, C . J. Mokeler, and P. A. Ullucci, Anal. Chem., 2018 (1972). (28) J. J. Chisolm, personal communication, 1972. A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O . 4 , A P R I L 1973
0
40, 44,
795
t a b l e I. Methods for the Determination of Lead in Blood Volume of blood used
Method
Optical emission
1.0 mi
Anodic stripping
0.2 ml
Polarographic
20 9
Spectrophotometric
10 mi
Atomic absorption
5 ml
Atomic absorption
0.25 ml
Atomic absorption Atomic absorption
2-5 ml
Atomic absorption
5-10 ml
Atomic absorption, "Sampling Boat" (P-E)
1.0 ml (0.25 mi min)
Atomic absorption, "Solid Phase Sampler" (I.L.)
0.5 ml
Atomic absorption, "Delves Cup"
0.01 ml
Atomic fluorescence, flameless
0.002 mi
Atomic absorption, flameless
0.1 ml (0.01 ml min)
Atomic absorption, flameless
1 PI
Atomic absorption, flameless
0.04 ml
Notes
TCA precipitation of blood, supernatant placed
1 mi
on electrode Acid digestion, ash dissolved in HCI and run. R.S.D. 12% Acid digestion, dithizone extraction, back extraction in NH4CI and run Acid digestion, dithizorie extraction into CHC13 and absorption at 505 nm Protein precipitation, extraction into MI BK with APDC and aspiration of organic layer Same as above, much smaller volume for aspiration Direct extraction with APDC-M I BK-aspiration Protein ppt with HC104, supernatant filtered and aspirated Lead concentrated on anion-exchange resin and aspirated Protein precipitation, supernatant placed on Tantalum Boat dried and vaporized Direct extraction with APDC-MIBK, organic layer placed on boat dried, vaporized Direct analysis, sample dried, ashed with H 2 0 2 in nickel crucible, vaporized Direct analysis after dilution signal measured at one wavelength, background at another Direct extraction with APDC-M IBK, organic layer vaporized Dilution in Triton X-100 and direct analysis after background correction applied Sample dried in special cuvette, digested with
H202
I K Hz 'Pb
ABSORPTION
.5K HZ
-
I K Hz
t BKG
Figure 1. Schematic diagram of automatic background corrector
RESULTS AND DISCUSSION Amount of Sample and Background Absorptions. The most significant problem in the course of our work involved background absorption during the atomization of lead in pyrolyzed blood samples. Because of the extreme volatility of lead, a rather low temperature must be employed during drying and pyrolysis of the sample. This leads to an incomplete pyrolysis of the blood which results in a large amount of white smoke during atomization. Analytical data indicate that the magnitude of the background absorption is quite significant in comparison to the actual lead signal as shown in Figure 2. In the figure, tracings are shown for lead in blood run according to the procedure described in the normal instrument mode and in the automatic background correction mode a t the 217.0 and 283.3 nm resonance lines, respectively. In the normal mode, the signal observed is due to lead plus any background absorption present. In the automatic background correction mode, only the true lead signal is observed. A comparison of the signals reveals that of the total signal 796
0
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
observed in the normal mode, about 75% is background absorption and approximately 25% is signal due to lead. The other interesting feature of Figure 2 is that the background absorption is not reproducible a t all. The data indicate the importance of keeping the background to a minimum so that it remains in the working range of the background corrector. To accomplish this, only 2.5 pl of whole blood is used for one measurement in the procedure. Selection of Analytical Wavelength. The lead line a t 283.3 nm was chosen over the 217.0 nm line for two reasons. First, the background absorption is greater a t 217.0 nm and base-line noise levels were lower at 283.3 nm as compared to 217.0 nm. Second, the 283.3 nm lead line appears to have better linearity in working curve. The working curve is shown in Figure 3 for both wavelengths. A t 283.3 nm, the lead signal is linear to 1 x 10-9 g P b or the equivalent of 0.4 pg/ml of lead in blood sample. At the 217.0 nm line, linearity extends to a level of 5 x 10-10 g or 0.2 pg/ml of lead in a blood sample. A work-
Pb
283.3 n m
BACKGROUND COR RECTION MODE
-
BACKGROUND CORRECTION MODE
NORMAL MODE
NORMAL MODE
Figure 2. Relative absorbances for backgrounds and analytical lines
~~
~
~
~~
Table It. instrumental Parameters Channel A
Mode of operation Hollow cathode tube Lamp current Photomultiplier P.M. voltage Slit width
Wavelength
A - Ba P b ( I . L.
Channel B
Cat. No. 62927)
4-5 mA R372 620 V 320 p 283.3 n m
H2
Continuum Source
( I . L. Cat. No. 63490) 25 m A
(Automatic Background Correction)
A recorder with response time of 0.5 sec. or less on full scale is employed. Range 100 rnV/full scale. Parameters for Model 355 Flameless Sampler
Mode Purge gas Gas flow rate D R Y and PYROLYZE setting Drying and pyrolysis time A N A L Y Z E setting Height of measurement
Automatic Araon 7 I./min 5-7 60 seconds 8 2 mm
Table I II. Analysis of Reference Blood Samples ( p g / 1 0 0 ml) Present techniaued Sarnpie
A B
C D
Reference value"
17flh 17flc 46f3 44f1 86f2 79fl 112fl 109f5
1
2
3
4
17f2 17f3 16f3 17f3 44f2 38f3 44f3 46f2 85f3 86f2 8 4 f 3 864-3 108f2 107f4 108f4 106+3
" All the precisions given are standard deviations.
* Flame AA Technique.
Spectrophotometric (dithizone). Flameless AA Technique.
ing curve is not necessary as long as analyses are conducted within this range with standards. Precision and Accuracy. Four standards of blood lead a t the different concentration levels were obtained from Dr. J. Julian Chisolm at Baltimore City Hospital, Baltimore, Md. The blood samples were previously spiked with known amounts of lead and digested. The lead content of
Pb :0 1
Figure 3. Calibration curves for lead at 217.0 and 283.3 nm
the samples was established by a number of techniques including conventional flame aspiration atomic absorption. Analytical results are in good agreement between theirs and ours. This indicates that blood samples can be directly analyzed for lead by the present technique. The samples have been found to be stable a t least six months. Table 111 indicates the precision of the technique as well as the accuracy obtainable. In our laboratory, these standards were analyzed by the standard addition technique on four different dates. Precisions are given in terms of the standard deviations. ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 4, A P R I L 1973 e 797
In summary, this technique has proved to be simple yet rapid as well as accurate and precise, Once the proper parameters are set, a t least 15-20 samples can be analyzed in one hour including standards. A blood sample volume of 10 ~ 1 easily , obtainable by a finger puncture, would be
sufficient for triplicate determination. All of these advantages yield a method which would be extremely valuable in a mass screening program involving young children. Received for review June 12, 1972. Accepted December 4, 1972.
Reaction of Lanthanum with Reagent Arsenazo-p-NO, N. U. Perigic-Janjic, A. A. Muk, and V. D. Canic Faculty of Technology, Novi Sad and Boris KidriE institute of Nuclear Sciences, Beograd, Yugoslavia
Of the many organic reagents, arsenazo 111, owing to its high sensitivity, has been extensively used for spectrophotometric determination of the rare earths (1-3). Further examination of mono- and bis-azo derivatives of chromotropic acid, particularly reagents with o-arsono-ooxy-azo functional groups, has shown that these reagents, besides arsenazo 111, may be used for the spectrophotometric determination of the rare earths (4-7). When the relationships between the protonation constant, hydrolysis of metal ions, medium acidity of the complexation, and molar absorptivity of the metal complexes and of the protonated reagent were studied, it was concluded (3-6) that the reaction of arsenazo-p-NO2 with lanthanum is very sensitive and selective comparable to that of lanthanum with arsenazo I11 ( 1 ) and arsenazo M (2, 6, 7 ) . The reagent arsenazo-p-NO,, besides having a oAs03H2 group in one benzene ring, has an -NO2 group in the paraposition in the other benzene ring.
In connection with its acidic characteristics, the p-NOz group has an effect on the acidity of this reagent similar to that of o-As03Hz in arsenazo I11 or m-SO3H in arsenazo M ff5, 6), i. e . , increases the acidity of the reagent. Preliminary studies of the reaction of arsenazo-p-NOz and lanthanum (5, 6) have shown that two types of complexes are formed: one with a maximum a t 650 nm, ratio La:R = 1:l and the other with a maximum a t 720 nm, ratio La:R = 1:2, probably. As is known, the reaction of lanthanum with arsenazo I11 ( 1 ) and arsenazo M (5, 6) does not give complexes identified by maximum a t 720 nm, thus making the lanthanumlarsenazo-p-NO2 reaction worth investigation. The long wavelength maximum a t 720 nm, would correspond to complexes of, so called, type 111, formed by the reactions of some other metals with the para substituents in the benzene rings of bis-azo derivatives of chromotropic acid (8-11). S. B. Savvin and A . A . M u k , Bull. Boris KidriE lnst. Nucl. Sci., 12, 97 (1961).
S. B. Savvin, "Arsenazo 1 1 1 . " Atornizdat, Moskva. 1966. Red D. I . Ryabchikov, "Sovrernennie rnetody anaiiza," Izd. "Nauka" Moskva, 1965, p 123. A. M u k and R. Radosavljevic, Croat. Chem. Acta, 39, 1 (1967). A. M u k and S. 8 . Savvin, Anal. Chim. Acta, 44, 59 (1969). A. M u k . S. 8. Savvin, and R. P. Propistzova, Zh. Anal. Khim.. 23,
127i'(1968). S. 8. Savvin. R. F. Propistzova, and R . V. Strelnikova, Zh. Anal. Khim., 24, 31 (1969). S. B.SavvinandT. V . Petrova, Zh. Anal. Khim., 24, 177 (1969) T . V. Petrova and S. B. Savvin, Zh. Anal. Khim., 24, 490 (1969). T. V. Petrova, N. Hakirnkhodzhaev. and S. B. Savvin, I z v . Akad. Nauk SSSR, Ser. Khim., 1970, 259. J. A. Perez-Bustamante and F. Burriel-Marfi. Anal. Chim. Acta. 51, 277 (1 970). 0
A N A L Y T I C A L CHEMISTRY, VOL. 45,
NO. 4,
APRIL 1973
For better understanding of the lanthanum/arsenazop-NO2 reaction, it is necessary to first study the reagent itself. The protonation constant of this reagent was studied in our previous work (5, 6).
EXPERIMENTAL R e a g e n t s a n d a p p a r a t u s . A standard procedure was used to synthesize arsenazo-p-NOz. The purity of the sample, found to be 70% calculated to the acid, was determined using potentiometric titration. The standard solution of lanthanum, with acidity about 0.1N was obtained by dissolving La203 in HC1. The lanthanum assay was determined gravimetrically as Laz03. The perchloric acid used was p.a. 70% "Kemika" and the sulfuric acid was p.a. 97% "Kemika." The spectrophotometers used were a recording Beckman DK-1A spectrophotometer and a Beckman Model DU spectrophotometer with 10.0-mm cells. Acetate, phosphate, and glycine buffers were used to maintain the pH of the solution. A Beckman Model Zeromatic p H meter was used for the pH measurements. S p e c t r a of Arsenazo-p-NOz. Arenazo-p-NO2 in slightly acidic solution (from 1Rr to pH 6) has a n absorption spectrum with a maximum at 545 n m , similar to the spectrum of arsenazo 111 in the same medium. Upon increasing the pH from 1 to 6, dissociation of sulfo and arson0 groups occurs. but the absorption spectrum of the reagent remains unchanged. With further pH increase, dissociation of OH groups from the naphthalene ring takes place, causing a change in s-electron configuration of the reagent, which in turn affects the absorption spectrum. In order to observe these changes, the absorption spectra at various acidities were studied. As the d a t a of Figure 1 show, the absorption maximum at pH about 8 is shifted toward longer wavelength (580 n m ) . This can be attributed to dissociation of the first OH group of the naphthalene ring. Determination of Dissociation Constant. If the dissociation of the first OH group of the naphthalene ring is expressed by the equation:
where HR is the ionic form of the reagent in which -S03H and -As03H2 acidic groups are already dissociated and R is the ionic form of the reagent in which the first OH group from naphthalene ring is dissociated, the dissociation constant may be calculated by the relation:
where (R--) and ( H R ) are the concentrations of corresponding ionic forms of the reagent and K , the dissociation constant. The concentration ratio of two ionic forms was determined from spectrophotometric measurements of absorbance at a given wavelength (545 n m ) by the known relation:
(R-) --
(HR)
A - Am As-A
(3)
