Document mmgarjL962vYQE4vpGZr0aVeB

: t : ! v f.'j* i.:. ; t< a. Jj ( 'i <>r-. \:i- ;; i'ii .) utu .A 1M IS W'Vrti ItIM,. (-*) t rra.^llif fi`om -3 iunii*, \ ions. isfoni uus.*i (hie Mt'lVV h tin* 12). Hoc.. I*, 37. Kn u . *7). / uusro&temniuon ol leal in Biological iK s{ riai With Dithissone Extraction at High pH JACOB CIIOLAK, DONALD M. IIUBBARD, AND ROLAND K. ltl liKKV Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Oititt The use of a procedure for the quantitative extraction f lead with dithizone at high pll lias facilitated tlie determination of lead in biological material. The high pll extraction step has been incorporated in the final estimation step of the Buinhach-Burkey method (1). The difficulties and advantages of the revised procedure are discussed and until) tical data are given to prove the udrqnutr nature of the revised procedure. L PROCEDURE for t.lie quantitative extraction of lead with dithizone at high pH, which was recently reported by Sny der (.(), has facilitated the determination of lead in biological mas e-rial. Following a personal communication early in 1945 (5), the jueci'duro was incorporated in the Bambach and Burkey method Ij and has been used satisfactorily since then for the routine analysis of idl types 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 extrac- :i;:u procedure to the analysis of biological material. For the o.nveniencc of analysis, the revised procedure is described step- wise, but only in so far as it, differs from that given by Bambach and Burkey (/). PROCEDURE Samples are prepared without change, and only the following riianges ill reagents are required. Ammonium Hydroxide-Potassium Cyanide Mixture. Dis itlvr 10 grams of potassium cyanide in 1000 ml. of ammonium hydroxide (specific gravity 0.900}. Lead-free ammonium hy droxide of the proper specific gravity may be made by passing tank ammonia into distilled water immersed in an ice bath. Dithizone Extraction Solution. Only one solution is needed-- dll mg. of Kastman dithizone per liter of lead-free (redistilled mid stabilized) chloroform. Isolation of Lead and Removal of Bismuth Interference. The iiiitial isolation of lead is carried out exactly as described by Uatnliaeh and Burkey (J). Because it is no longer necessary to lisrrvo each 5-ml. extraction in order to fit the samples into the three coiieenI rat ion ranges used by Bambach and Burlcoy (I), tiie extraction may be started with 10, 15, or 20 ml. of dithizone solution as the analyst prefers. The approximate total quantity nf lead, however, must be known and this is determined from the total volume of extraction solut ion used. Each 5 ml. of dithizone extraction solution will extract approximately 40 micrograms of bail (f). 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 to or below 150 mierograms before adding the 50 ml. of the pH 3.4 buffer which is used t i> separate the lead from the bismuth. Final Estimation of Lead. To the 50 ml. of pH 3.4 buffer (1) containing less than 150 mierograms, add 20 ml. of the amiiuiuittm hydroxide-potassium cyanide mixture. (The automatic pipet shown in Figure 1 has been found convenient for this purpose. It was designed and made in this laboratory by W. J. Votuikcr.) Add 15 ml. of dithizone extraction solution and shake for t minute, releasing the pressure which develops through the stopper rather than through the stopcock. The colors in an en tire series are developetl in this way before proceeding to the pliiiiumetry. Photometry. Plata; a small pledget of cotton in the tip of Hie stem of efteh funnel, and just before filling the cells, discard `1 to 3 ml. of the chloroform phase by allowing it to pass through die cotton pledget. Fill the coll by filtering the chloroform -elution through the pledget and read the density or the transtmllancy of the solution at 510 mg 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 io make measurements with only two cells, a cell of 50- mm. light, path for quantities from 0 to 15 mierograms and a cell of 10-min. light path for quantities from about 15 to 100 or 110 mierograms of lead. (A cell with a 5-mm. light path must be used when the amount of lead is between 110 and 150 mierograms.) 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. In Table I are listed typical results chosen at random from ap proximately one hundred samples, duplicate aliquots of whieli were analyzed in parallel by the Bambach ami Burkey {/) method and the modified procedure. In the case of the blood samples, these two sei of resells are compared with the spectrographic finding.-:. The. comparative de grees of accuracy and re producibility of results ol gained on two series of samples by the old and new procedures are indi cated in Table II. The ash of a series of samples of feces, in the one ease, and of urine, in the other, which remained follow ing certain other experi mental observations, was composited and then divided into 40 equiva lent samples, respec tively, for the parallel analyses. The other parallel series eim-isled of two sets of samples of synthetic urine l.J) u> which known amounts of lead were added. Figure 1. Automatic Dispensing Pipet DISCUSSION Inspection of Tables I and II shows that there is little difference ill the findings obtained by the two methods. Tin- dif ferences between tin- re spect ivo moan values in Table II, however, mo 671 N36883 DUP050311839 672 J Tabic f. Results Obtained on Duplicate Samples by Extracting with Dithizonc at pH 9.5 and 11.5 Description (Trine 1JS5 l rino 12S2 [ rine 12!)!) brine 1487 Found byBamhnch and Burkey Mothod (J) Mg. 0.325 0.175 0.160 0.100 Found by Revised Procedure Wff. 0.320 0.177 O.lfiO 0.187 Fores 1210 Feces 143S Feces 1442 Feces 1368 0.430 `0.57 0.52 0.31 0.430 0.50 0.51 0.33 Food (mixed 24 hour total) 1369 Food (mixed 24 hour total) 1421 Food (mixed 24 hour total) 1576 Food (mixed 24 hour total) 1624 0.19 0.18 0.16 0.33 0.10 0.10 O.in 0.33 Spcctrographic (2) ifo./too g. Mff./SOO g. Blood 2152 Blood 2153 Blood 2185 Blood 2102 0.050 0.060 0.060 0.055 0.055 0.066 0.044 0.061 0.051 0.066 0.044 0.001 statistically significant; slightly superior accuracy is shown by the. revised method, particularly in the higher ranges of concen tration. The reproducibility of the results, on the other hand, is somewhat superior in the case of the Bambach and Burkey method (7). In the analysis of biological material, special precautions must be taken to prevent interference by bismuth. For this reason, it is necessary to make a preliminary extraction, which isolates lead and bismuth from the extraneous ash, and then to remove the bismuth before proceeding to the final load step of extraction and photometry. The initial extraction may' be made within the range of pH S.o and 11.5, but it is best to work at the lower pH (not exceeding 9) if the bismuth separation is to be made by washing the initial extract with the buffer solution at pH 3.4 (1). Changes in the buffer at this point will result in low recoveries of lead, and the losses arc not reclaimed unless the initial chloro form extract is washed thoroughly with water to removo en trained alkali. When the initial extraction is made at pH 8.5, a single wash of tire chloroform extract with 50 ml. of distilled water is sufficient to remove all the entrained alkali. On the other hand, at pH 11.5, interfering quantities of alkali remain even if the chloroform extract, is washed with two 50-mi. portions of distilled water, and there may be significant losses of lead. Hence, pH S.5 is chosen for the initial extraction. Tabic If. Reproducibility and Accuracy of Two Analytical Procedures Applied in Parallel , Material 1NSJon. rotff Analyses Method -Micrograms of Lead--. -Std*. ~St' d*. Mean error dev. Ashed feces, composited and subdivided Ashed urine, compositedi and subdivided Synthetic urine, 8 micro>grains Pb added Synthetic urine, 40 micrograins Pb added 20 20 20 20 10 10 10 10 Bambach 42.63 and Burkey Revised 44.00 Bambach 8.69 and Burkey Revised S.90 Bambach 7.9 and Burkey Revised 8.16 Bambach 38.66 and Burkey Revised 39.8 0.064 0.123 =0.056 0.050 0.024 0.073 0.209 0.201 0.29 0.55 0.250 0.224 0.077 0.230 0.66 0.64 The extraction at high pH possesses a distinct advantage when carried out in the presence of moderate amounts of phos phate ion. This fact is indicated in Tabic III, wherein are re corded the results obtained in the analysis of samples containing 50 micrograms of lead buffered at pH 3,4, to which various amounts of phosphate ion were added, following extractions at pH 9.5 and pH. 11.5, respectively. Extractions with dithizone were made af ANALYTICAL CHEMISTRY ter the phosphate and lead had linen allowed to remain in contact for various periods of time. The extraction of lead with dithizonc at pH 11.5 is quantitative even in the presence of 5 mg. of phos phate ion. On the other hand, low recoveries are obtained at pH 9.5 in the presence of 1 mg. of phbsphaLe or less. Actually, other experiments have shown that quantities of phosphate as low as 0.5 mg. in the final solution prevent the.complete extraction of lead. In the Bambach-Burkoy meihod (7) solutions of lead (iitiiizonatemaybe subjected to photometry without preliminary fillration. Although the same procedure may be followed in exl meting at- high pH, the results so obtained show a leaser degree of repro ducibility than do those obtained after filtration. A convenient and rapid method of filtration is provided by the insertion of a small pledget of cotton into the stem of the funnel. Xo special purification of the cotton is required other than that accomplished by wasting 2 ml. of the chloroform phase through the pledget be fore filtering the remainder into the measuring ceil. The use of a specific type of cell (Stylo D, American Instrument Company, or equivalent) permits the employment of small vol umes of solution and results in an increased sensitivity of detec tion. Only 15 ml. are used and only 10 ml. of this amount are required to fill the cell (having a light path of 50 mm.) which is used in the range 0 to 15 micrograms of lead. Table III. Effect of Phosphate Ion on Extraction of Lead with Dithizonc (50 micrograms of Pb) Added P0<~ Mg. 0 1 2 3 4 5 pH of Final Extraction 9.5 11.5 9.5 11.5 9.5 11.5 9.3 11.5 11.5 11.5 Extracted Immediately 7 50 49 33 50 48* 51 49.8 50 Extracted after 10 mitu 20 min. 30 min. 7 77 50 49.9 42.5 49.7 10.5 49 8.5 50 49 50 48 4..9 As shown by Snyder (-(), it is no longer necessary to prepare separate standard solutions of dithizone for the three ranges of lead concentration, and the same extraction solution which is used for the initial extraction can be used in the final estimation of color. For the final extraction of lead, the proper quantity of di thizone also may be included in the ammonium hydroxide-potas sium cyanide mixture. After adding the mixture, it is only nec essary to add 15 ml. of clear chloroform to extract the lead. Such a mixture has been employed in a rapid screening test for lead in. urine (S). Solutions of this type have been kept apparently un changed for more than six months. The great convenience asso ciated with the stability of such solutions is obvious, especially in laboratories in which only occasional lead determinations arc carried out. Under these conditions, the buffer solution contain ing the dithizone can be stored in a refrigerator, and preserved further in an unchanged state. The initial extraction solution can then be made up as required and in sufficient amounts to take eare of current samples, s LITERATURE CITED (1) Bambach, K., and Burkey, R. E., Ism. En g . Ch e m., An a l . Ed .. 14, 904 (1942). (2) Cholak, J.,/ifd,, 7.2S7 (1935). (3) Cholak, J., Hubbard, D. M., and Burkey, R. E., J. Ind. Hyo- Toxicol., 30, 59 (194S). (4) Snyder, L. J., An a t .. Ch e m., 19, GS4 (1947). (5) Snyder, L. J., Ethyl Corp., Baton Rouge, La., personal communi cation, 1945. Re c e iv e !. November 7, 1047. DUP050311840