Microdetermination of lead in Biological Material With Dithizone Extraction ut High p H J4COB CHOLAK, DONALD 41. HUBBARD, AND ROL.IND E. BLRKEY Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio The use of a procedure for the quantitative extraction of lead with dithiAone a t high pH has facilitated the determination of lead i n biological material. The high pH extraction step has been incorporated i n the final estimation step of t h e Bambach-Burke! method ( I ) . The difficulties and advantages of the revised procedure are discussed and anal>tical data are given to prove the adequate nature of the revised procedure.
A
nini. light path for quantities from 0 to 15 micrograms and a cell of 10-mm. light path for quantities from about 15 to 100 or 110 micrograms of lead. (A cell with a 5-mm. light path must be used when the amount of lead is between 110 and 150 micrograms.) The working curves are prepared from known amounts of lead added to the pH 3.4 buffer. A blank, starting with the pH 3.4 buffer, is run in order to obtain the daily zero points of the calibration curves. A blank of all of the reagents used in the complete extraction must also be determined and subtracted from the values read from the calibration curves.
PROCEDURE for the quantitative extract,ion of lead a i t h
dithizone at, high pH, which was recently reported by Snyder ( d ) , has facilitated the deterniinat’ionof lead in biological material. Following a personal communication early in 1945 (@, the procedure was incorporated in the Bainbach and Burkey method (f) and has been used satisfactorily since t,hen for t’he routine analysis of all t,ypes of samples. The revised procedure represents a significant advance in analytical technique, and is deemed worthy of presentation in order to point out the difficulties and advantages encountered in the application of the high pH extraction procedure to the analysis of biological material. For the convenience of analysts, the revised procedure is described stepwise, but only in so far as it differ8 froin t h a t given by Banibach and Burkey ( 1 ) .
RESULTS
In Table I are listed typical results chosen ctt random from approximately one hundred samples, duplicate aliquots of which were analyzed in parallel by the Banibach and Burkey (1) method and the modified procrdure. In the ( m e of the blood samples, these two sets of results are compared with the apectrographic findings. The comparative degrees of accuracy and reproducibility of results obtained on two series of samples by the old and ne\T procedures are indirated in Table IT. The ash of a series of samples of feces, in the one case, and of urine, in the other, which remained following certain other experimental observations, was composited and then divided into 40 equivalent ,samples, rcspectively, for the parallel analyses. The other parallel series consisted of two sets of samples of synthetic urine ( 2 ) to Ivhieh knon-n amounts of lead were added.
PROCEDURE
Samples are prepared without change, and only the following changes in reagents are required. * Ammonium Hydroxide-Potassium Cyanide Mixture. Dissolve 10 grams of potassium cyanide in 1000 ml. of ammonium hydroxide (specific gravity 0,900). Lead-free ammonium hydroxide of t,he proper dpecific gravity may be made by passing tank ammonia into distilled nater immersed in an ice bath. Dithizone Extraction Solution, Only one solution is needed30 mg. of Eastman dithizone per liter of lead-free (redistilled and stabilized) chloroform. Isolation of Lead and Removal of Bismuth Interference. The initial isolation of lead is carried out exactly as described by Bambach and Burkey ( I ) . Because it is no longer necessary to observe each 5-ml. extraction in order to fit the samples into the three concentration ranges used by Bambach and Burkey ( I ) , the extraction may be started a i t h 10, 15, or 20 ml. of dithizone solution as the analyst prefers. The approximate total quantity of lead, homever, must be known and this is determined from the total volume of extraction solution used. Each 5 ml. of dithizone extraction solution will extract approximately 40 micrograms of lead (1). If the quantity of the dithizone extraction solution used indicates the presence of more than 150 micrograms of lead, an aliquot is removed to bring the quantity of lead t,o or be1o-x 150 microgranis before addin the 50 ml. of the pH 3.4 buffer which is used to separate the l e a d h m the bismuth Final Estimation of Lead. To the 5 0 11x1. of pII 3.4 buffer i f ) containing less than 150 micrograms, add 20 ml. of the ammoniuin liydroxide-potassium cyanide mixture. (The automatic pipet s h o m in Figure 1 has been found convenient for this purpose. I t was designed and made in this laboratory by TI-. J. I-ounkcr.) Add 15 ml. of dithizone extraction solution and shake for 1 minute, releasing the pressure which develops through the 6toppcr rather than through the stopcock. The colors in an entire series are developed in this way hefore proceeding to the photometry. Photometry. Place a small pledget of cotton in the tip of the stem of each funnel, and just before filling the cells, discard 2 to 3 ml. of the chloroform phase by allon-ing it to pass through the cotto11 pledget. Fill the cell hy filtwing the chloroform solution through the pledget and read the density or the transmittancy of the solution a t 510 mp in any suitable photometer. The proper size of cell to use is determined as in Snyder’s method ($1, from the depth of color in the chloroform phase. It is usually possible to make measurements with only two cells, a cell of 50-
DISCUSSION
p i p e t , filler
bore i n
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Figure 1. lutoniatic Dispensing Pipet
671
Inspection of Tables I and I1 shows that there is little difference in the findings obtained by the two methods. The differences between the respective mean values in Table 11, holTever, are
.
ANALYTICAL CHEMISTRY
672 Tahle I. Results Obtained on Duplicate Samples h? Extracting with Dithizone a t pH 9.5 and 11.5 Found by Bambach and Burkey Method ( 1 )
Description Crine 1185 L-rine 1282 Crine 1299 Urine 1487 Feces 1216 Feces 1438 Feces 1442 Feces 1368 Food (mixed Food (mixed I'ood (mixed Food (mixed Blood Blood Blood Blood
21.52 2133 2185 2192
0,325 0.175 0,160 0.190
24 24 24 24
hour hour hour hour
total) total) total) total)
0.430 0.57 0.52 0.31 0.19 0.18 0.16 0.33
1369 1421 1576 1624
Spectrographic (9) .21g./100 a. 0.050 0.0.35 0,060 0.066 0.050 0.044 0,055 0.001
Found h y Revised Procediire
0.320 0,177 0.160 0.187 0.430 0.56 0.51 0.33 0 10 0 19
0 10
0.33 .Ilu.
'IOIl
u.
0.051 0.066 0.044 0.061
ter the phosphate and lead had been allowed to remain iii contact for various periods of time. The extraction of lead with dithizone a t pH 11.5 is quantitative even in the presence of 5 mg. of phosphate ion. On the other hand, lo^ recoveries are obtained at, pH 9.5 in the presence of 1 mg. of phosphate or less. Actually, other experiments have shown that quantities of phosphate as lox as 0.5 mg. in the final solut,ionprevent the complete extraction of lead. In the Bambach-Burkey method ( 1 ) solutions of lead dithizonatc may be subjected to photonietry without preliminary filtration. AUthoughthe same procedure may he folloned in extracting at high pII, the results so obtained shoir- a lesser degree of reproducibility than do those obtained after filtration. A convenient and rapid method of filtration is provided by the inwrtion of a small plrdgct of cotton into the stcni of the funnrl. S o special purification of the cotton is required other than that acromplished by wasting 2 ml. of the chloroform pha7e through thta pltvlget hefore filtering the remainder into the nieaeuring ~ 1 1 . The use of a specific type of cell (Style D, hnierican Instrument Company, or equivalent) permits the employment of small volumes of solution and results in an increased sensitivity of detertion. Only 15 nil. are used and only 10 ml. of t,his amount ar? required to fill the cell (having a light path of 50 mm.) n-hich i.q used in t h t range 0 to 15 microgram$ of lead.
statistically significant; slightly superior accuracy is shown by the revised method, particularly in the higher ranges of concentration. The reproducibility of the results, on the other hand, is somen-hat superior in thc case of the Ranihach and Rurkey method ( 1 ) . 'I'ahle 111. Effect of Phosphate Ion on Extraction of Lead Tn thr analysis of biological niaterial, special precautions must with nithizone be taken to prevent interference by bismuth. For this reason, it (ij0 microgrnin- of Pt)) is necessary to make a preliminary extraction, n-hich isolates p H uf Extracted after ddded Final Extrarted lead and bismut,h from the extraneous ash, and then to remove POIExtraction Iminediatrly 10 min. 20 min. 30 min. the bismuth before procerding to the final lead step of rstraction .If?. Y Y Y Y and photometry. Tlie initial extraction may be made within 50 30 9.5 .. 11.5 49 49.9 the range of pH 8.5 and 11.5, h i t it is beet to n-ork at the Ion-er .. .. 9.5 33 42.5 pH (not exceeding 9) if thrs tiimiuth ueparatioii is to be made by .. .. .. 11 5 49.7 50 . . 9 . 5 10.6. washing the initial extract vith the huffer solution at pH 3.4 ( 1 ) . .. 11.5 48 49 Changes in the buffer at this point xi11 result in I o n rwovcries .. .. 0.5 8.5 . . . . 11.5 51 50 of lead, and the losses are not reclaimed unless t h v initid chloro48 49 11.5 49.8 49 50 11.5 50 .. *. form extract, is ~ a s h e dthoroughly n-ith water to remove entrained alkali. Khen the initial ext,raction is macle a t p H 8.5, a single wash of the chloroforni estract with 50 nil. of tiiatillcd water is sufficient to remove all the entrained alkali. 0 1 1 thv As elio\vri Iiy Snyder ( d ) , it is 110 longer necessary to prepare other hand, a t pH 11.5, interfering quantitips of alkali i~cniai~i separatr standard solutions of tlithizone for the three ranges of even if the chloroform extract is washed with two 50-nil. portions lead ronc*t.ntration,and thts same extraction solution which is used of distilled n-ater, and t,hc>rramay be significant 1c1 for the initial extraction can be used in the final estimation of Hence, pH 8.5 is chosen for thc initial extraction. color. For the final estraction of lead, t,he proper quantity of dithizoric. also may be included in the ammonium hydroxide-potassium cyanide mixture. hfter adding the mixture, it is only ne?Tahle 11. Reproducihilit? and Accuracy of Two .inalytical essary to add 15 ml. of clear chloroform to extract the lead. Surh Procedures 4pplied in Parallel a mist,ure has been employed in a rapid screening test for lead in -Micropranis of Leadurine (3). Solutions of this type have been kept' apparently unKO. of Std. Std. changed for more than six months. The great convenience assoAlaterial Anal) .PS Method Mean error dev, ciated \\-ith the stability of such solutions is obvious, especially Ashed feces, comDosited 20 Raniharh 42.63 10.064 1 0 . 2 9 a n d subdivided and Burkey in laI)oi,atories in which only occasional lead determinations are 44.00 1 0 . 1 2 3 f0.35 20 Krviied Ashed urine. comDosited 20 Aainbaclr 8 . 6 9 10.086 1 0 . 2 6 0 carriI,, a n d B u r k e y , R. E., J . I n d . H u g .
To+icol., 30, 59 (1948). (4) S n y d e r , L. J., AN.AL.CHEM.,19, 6 8 4 (1947). ( 5 ) S n y d e r , L. J., E t h y l C o r p . , B a t o n R o u g e , L a . , p e r s o n a l c a t i o n , 1945.
E E C E I V E D November 7, 1947.
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