Microdetermination of Lead in Blood by Flameless Atomic Absorption Spectrometry Jae Y. Hwang, Paul A. Ullucci, Stanley B. Smith, Jr., and Arthur L. Malenfant Instrumentation Laboratory Inc., 113 Hartwell Avenue, Lexington, Mass. 021 73
A MICROANALYTICAL METHOD for the determination of lead in blood utilizing a flameless atomic absorption technique has been developed. This technique employs electrothermal heating of the sample in an enclosed cell to produce atomic lead vapor. The atomization takes place in an argon atmosphere that not only increases the atomic population of the analyte, but also retards the oxidation of the tantalum strip that serves as the sample boat. The technique is a modification of that described by Donega and Burgess ( I ) . A high speed strip chart recorder for absorption signals has been substituted for the oscilloscope, and an automatic background correction system has been added to the basic absorption unit used in their experiment. The blood sample is treated prior to analysis by chelation and extraction of lead with ammonium 1-pyrrolidinecarbodithioate (APDC) and methyl isobutyl ketone (MIBK), respectively. This scheme of sample preparation was recently reported by Farrelly and Pybus (2). A minor variation in their formula was made in preparing the sample for analysis by the flameless atomic absorption technique. The solvent extraction is accomplished directly from whole blood samples and requires neither precipitation of the protein nor adjustment of the blood pH. This is significantly faster than the conventional technique, since it eliminates the time required for the precipitation of protein with nitric, perchloric, or trichloroacetic acids. The principal arguments for the adoption of this modified technique are that first, it avoids the unsatisfactory results experienced with the direct application of the blood sample : the excessive background introduced by the matrix and the buildup of residue. Second, it avoids the introduction of corrosive reagents. The acids required in the protein nitric, precipitation technique, 10 % trichloroacetic, 30 and 7 x perchloric, gave significantly high blank values Third, the solvent extraction technique provides certain advantages, e.g., the shorter solvent evaporation time permits a more rapid analysis, the process is free of chemical and physical interferences, there is no accumulation of residue on the tantalum strip, and the method is effective with very small blood samples, of the order of 100 pl. EXPERIMENTAL
The absorption cell is molded of Lexan, with a quartz window at each end. It is equipped with an inlet and an outlet for the argon gas, with a sample port, and with electrodes to which the tantalum strip is attached. A schematic diagram of the cell appears in Figure 1. The argon gas system consists of a standard argon tank and a two-stage regulator, which is fitted with a shut-off valve and a flow meter (No. RMA-fj-SSB, F. W. Dwyer Mfg. Co., Michigan City, Ind.). The combination of a Variac (Type 2 pf. 1010, Staco, Inc., Dayton, Ohio) and a step-down transformer (Type BB, Dongan Electric Mfg. Co., Detroit, Mich.) is (1) H. M. Donega and T. E. Burgess, ANAL.CHEM.,42, 1521 (1970). (2) R. 0. Farrelly and J. Pybus, Cliri. Chem., 15, 566 (1969).
Figure 1. Schematic diagram of the absorption cell used to provide electric current to heat the tantalum strip. The strip has a V-shape indentation in its center which can hold between 50 and 150 p1 of sample. All atomic absorption measurements were made with an Instrumentation Laboratory Model 353 dual double-beam atomic absorption spectrophotometer. The design features of this instrument have been outlined in the literature (3). Automatic background correction is carried out according to the Operations Manual Reagents. The following solutions were used in this study: Formamide, APDC 2 x solution, Saponin 1 solution, and water-saturated MIBK. Standard solutions of lead nitrate containing 0.1,0.3, and 0.5 pg/ml of lead were prepared immediately before use from IL standard No. 36820, a solution of 1000 pg/lead per ml. All solutions were prepared using only distilled, deionized water. Procedures. SAMPLE PREPARATION. To 0.1 ml of whole blood were added, in order, one drop of 1% Saponin solution, 0.2 ml of Formamide, 0.1 ml of 2% APDC, 0.5 ml of water saturated MIBK. The blank and standards, prepared from water and standard solution, respectively, were measured and treated by the same reagents as the sample of whole blood. The Saponin acts to hemolyze the blood while the formamide solution prevents emulsion. The sample is mixed following the addition of each reagent, and is then mixed thoroughly with MIBK. The aqueous and organic layers separate completely without centrifugation. At this (3) S . B. Smith, Jr., J. A. Blasi, and F. J. Feldman, ANAL.CHEM., 40, 1525 (1968).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10,AUGUST 1971
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I
WrlHOUT BPLKQROUND CORRECTION
1
/ r
30 / '
IS
IS
TIME
WH W Q R O U N D CORRECTION
I
Pb
It
so
IS
30
uc.
I
IS
30
TIME n c
Figure 2. Actual recorder tracings
Table 1. Instrumental Parameters Channel A Channel B Hollow cathode Lead 62927 HQ continuum 63490 Lamp current 5 mA 25 mA Photomultiplier R106 P.M. voltage 530 V Slit width 320 p (spectral bandpass 0.8 nm) Wavelength 217.0 nm Table 11. Recovery Study Unspiked Sample + sample, 10 r g ZPb, Sample A
B C
rg
z
39 32 28
z
Recovery,
z
47
95
42
100
39
104
point, the samples are ready for the absorption measurements. MAKINGTHE DETERMINATIONS. Twenty microliters of the MIBK extract of the sample are placed on the tantalum strip by a disposable micropipet (Scientific Products Co., No. P4518-20) which gives h 0 . 5 Z of sampling reproducibility. The Variac is set initially at approximately 7 volts, passing a current of 17 amperes to evaporate the organic solvent. Argon gas is flushed continuously through the absorption cell at 7.5 standard cubic feet per hour (SCFH). In approximately 30 seconds, when the strip is dry, the Variac source is turned off and the dial is set to 24 volts in preparation for the lead measurements. The high current, 64 amperes, passed at this Variac setting volatilizes the sample and produces an immediate response by the photometer which appears as a sharp spike on the recorder. Upon the completion of the run, the Variac is immediately turned off; the system is ready for the next determination. ALIGNINGTHE INSTRUMENT. Optimum performance of the instrument requires that the absorption cell, the mono1320
chromator, and the tantalum strip be placed in exact alignment. The absorption cell is visually aligned with the optical path. A narrow cardboard strip is placed through the sample port and allowed to rest on the tantalum strip. The cell is then adjusted so that the hollow cathode light beam is approximately 2 to 3 mm above the center of the tantalum strip. The alignment and geometry of the tantalum strip are extremely critical in attaining maximum sensitivity. Other operating specifications are presented in Table I. AUTOMATIC BACKGROUND CORRECTION.Because of their background and residue buildup characteristics, direct blood samples were abandoned in favor of MIBK extraction of PB-APDC. Background effects from blood samples and from standard solutions were investigated after extraction. The actual tracings of the spectra of both blood and standard solutions, with and without background correction, are presented in Figure 2. These spectra are shown as functions of sensitivity in milliabsorbance units (mA) and time in seconds. In the tracings representing measurements without background correction, the processes of the evaporation of MIBK and the pyrolysis of Pb-APDC appear as double peaks. Pb absorbance signals are generated as soon as high currents are applied to the tantalum boat in the absorption chambers. With automatic background correction, the amplitude of the broad peak is greatly diminished. The undulating spectrum preceding the lead peak indicates that there is in sequence an undercorrection of background in excessive evaporation of MIBK, overcorrection of background when evaporation ceases, and finally undercorrection of background in the pyrolysis process. The amplitude of the lead peaks is the same with or without background correction. This indicates that APDC is completely pyrolyzed even at a later stage of evaporation of MIBK under lower current. For this reason, background correction was found unnecessary in the analysis. ANALYTICAL RESULTS
A recovery study was made for samples A, B, and C in triplicate runs (Table 11). The analytical results of this study were compared with those obtained by two different
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
techniques of atomic absorption spectrometry. One analysis was done by the conventional nebulizer-flame absorption, the other by the solid phase sampler technique which is equivalent to the sampling boat technique (4). The conventional technique calls for the precipitation of serum protein with trichloroacetic acid prior to the measurement of lead absorbance. The solid phase sampler technique calls for MIBK extraction of Pb-APDC and volatilization in a sampler vessel by an air-acetylene flame. A study of Table I11 shows good agreement among the results of the three different techniques with no significant error patterns.. In comparison with the direct analysis of lead using heated graphite atomizer or carbon rod atomizer (3, the present technique, when coupled with a solvent extraction technique, has a few advantages, i.e., no chance of losing lead during pyrolysis process of blood samples, ease of preparing standards, reduction or elimination of chemical interferences, and no residue buildup or background absorption from blood samples. However, on samples from patients on EDTA or penicillamine therapy, recoveiy of lead is incomplete. As Farrelly and Pybus pointed out (2), perchloric acid digestion is necessary if accurate results are to be obtained.
(4) J. Y. Hwang, P. A. Ullucci, A. L. Malenfant, I.L. Reprint, Atomic Absorption Spectrometric Determination of Lead in Blood by the Solid Phase Sampler Technique (1970). (5) M. D. Amos, P. A. Bennett, K. G. Brodie, P. W. Y. Lung, and 43, 211 (1971). J. P. Matonsek, ANAL.CHEM.,
Table HI. Comparison of Various Atomic Absorption Methods (fig %) Flame AAa Flameless AAb SPS AA” 15 19 24
24 28 30 52
14 20 23 24 26 29 52
... 19 24 21 27 33
...
Conventional flame atomic absorption technique coupled with TCA protein precipitation method. Present technique combined with APDC-MIBK chelationextraction method. Solid phase sampler technique combined with APDC-MIBK chelation-extractionmethod. a
In summary, this technique has proved to be simple and gram very rapid. The precision of 5x RSD at 1.2 X level and the sensitivity of 7 X 1O-I’ gram for 1 % absorption signal or 4.4 milliabsorbance units can be attained. Because it is effective for the determination of lead in blood samples obtained by finger puncture, the method will be extremely valuable in mass screening programs involving young children. RECENEDfor review March 17, 1971. Accepted May 19, 1971. Excerpts from this paper were originally presented at the Northeastern Section of the American Chemical Society Meeting, October 1970.
Atomic Absorption Spectrometry of Copper with Selected Organic Solvents after Extraction from Aqueous Solution with 8-Hydroxyquinoline J. H. Culp, R. L. Windham, and R. D. Whealy Department of Chemistry, Texas A&M University, College Station, Texas 77841
THEUSE OF SOLVENT extraction techniques in atomic absorption spectrometry continues to increase as this method has proved to be a simple and convenient means of selectively extracting and concentrating a desired metal or group of metals while also affording enhancement of sensitivity. Allan ( I ) and Bode and Fabian ( 2 ) have reported on the use of several organic solvents in the determination of copper. Dagnall and West (3) have studied the use of organic solvents for the extraction of lead as well as the use of gasoline as a solvent for the determination of lead as well as the use of gasoline as a solvent for the determination of tetraethyl lead. The use of 8-hydroxyquinoline in solvent extraction is well documented ( 4 , 5).
Even though the use of organic solvents in atomic absorption spectrometry is becoming a more common technique for enhancement of sensitivity, the mechanism producing this enhancement is complicated and has not been fully explained (I, 6). Not all organic solvents produce enhancement of sensitivity. Many solvents while serving as good mediums for solvent extraction of metal chelates actually suppress the atomic absorption sensitivity as compared to the sensitivity of the metal in water. This study investigates the potential of some additional solvents to solvent extraction and their application to atomic absorption spectrometry. EXPERIMENTAL
(1) (2) (3) (4)
Apparatus. Absorption measurements were made on a Jarrell-Ash Series 82-360 atomic absorption/flame emission spectrometer system equipped with a HETCO total consump-
1957. (5) C. H. R. Gentry and L. G. Sherrington, Analyst, 75, 17 (1950).
(6) J. W. Robinson, “Atomic Absorption Spectroscopy,” Marcel Dekker, Inc., New York, 1966, Chap. V.
J. E. Allan, Spectrochim. Acra, 17, 467 (1961). H. Bode and H. Fabian, 2. Anal. Chern., 162, 328 (1958). R. M. Dagnall and T. S . West, Tularftu, 11, 1553 (1964). G. H. Morrison and H. Freiser, “Solvent Extraction in Analytical Chemistry,” John Wiley & Sons, Inc., New York, N. Y.,
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