August 15, 1941
ANALYTICAL EDITION
(3) Brown, J. B., and Frankel, J., J. A m . Chem. SOC.,60, 54 (1938). (4) Brown, J. B., and Stoner, G . G., Ibid., 5 9 , 3 (1937). ( 5 ) Chappuis, Trav. Mem. Bur. Int., XVI (1914). (6) Clevenger. J. F., IND. ENG.CHEM.,16, 854 (1924). (7) Dunbar, R. E., Ibid., Anal. Ed., 11, 516 (1939). (8) Farkas, A., and L., Ibid., 12, 296 (1940). (9) Hardy, W., Proc. Roy. SOC.(London), A112,47 (1926). (10) Hennig and Justi, 2.Instrumentenk., 50,343 (1929). (11) Hodgman, C. D., “Handbook of Chemistry and Physics”. 23rd ed., Cleveland, Chemical Rubber Publishing Co., 1939.
583
Keyes, J . Muth. Phus., 1, 242 (1922). Moran, T., Proc. Roy. Soc. (London), A118, 548 (1935). Niethammer, G., Mikrochemie, Neue Folge, 1, 223 (1929). Schmidt, E., 2. Ver. deut. Ing., 7 6 , 1025 (1932). (16) Shinowara, G. Y . ,and Brown. J. B., J . A m . Chem. Soc., 60, 2734 (1938). (17) Strong, J., “Procedures in Experimental Physics”, New York, Prentice Hall, 1938. (18) Zscheile, F. P., and White, J. W., IND.ENG.CHEM.,Anal. Ed., 12, 436 (1940). (12) (13) (14) (15)
Determination of Lead in Biological Material A Polarographic Method JACOB CHOLAK AND KARL BAMBACH Kettering Laboratory of Applied Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio
P
OLAROGRAPHIC methods for the determination of lead in biological material have been described by Shikata ( I d ) , Yoshida (15), Hamamoto ( 7 ) , Teisinger (IS), and Forche (6). Several of these methods can be applied only to specific materials (6, 7 , I d , IS), others lack sensitivity and accuracy (6, 15), and none is entirely satisfactory for general use. An effort to overcome some of these difficulties led to the development of a procedure for the direct electrolytic deposition of lead from solutions of ashed material (9)and made possible the quantitative isolation of lead in small volumes of test solution which can be polarized satisfactorily. A method based on this electrolytic technique has proved to be applicable to every type and condition of sample likely to be encountered. It is presented here not as an improvement over the best of the chemical and spectrographic methods in current use but as an interesting and successful application of a new analytical tool. I n addition to the electrolytic means of isolating and concentrating the lead, a method is also given for the separation of the lead with dithizone followed by electrolytic concentration, if necessary. The latter provides for the accurate analysis of certain samples (feces and individual or mixed foods) with greater speed and convenience.
Polarographic Technique Types of apparatus and the theory of analysis by the use of the dropping-mercury cathode have been fully covered by Hohn (8), Kolthoff and Lingane (IO), and Walkley (IC), among others; therefore we need concern ourselves only with the practical applications of the method. Although the operation of the equipment is relatively simple, a number of factors must be considered if the end result is to be reliable. Purity of reagents and precision in chemical processes are obviously required, and it should also be recognized that the current-voltage curves are markedly affected by the size of the mercury drop, its dropping rate, the temperature, and by the composition of the test solution. Several of these difficulties may be overcome by rigid standardization of technique, but, despite continual and tedious experimentation, standard procedures can scarcely be devised which will permit the use of a single working curve for every type of sample that may be encountered. Forche (6) has shown, however, that the employment of “internal standards”, an adaptation from spectrographic analysis, results in a marked stability of the working curves. He (5, 6) introduced a known amount of cadmium into his standards and samples, and then employed the ratlo of the lengths of the lead and cadmium steps as a measure of the lead concentration.
After the procedure had been standardized, the ratios were independent of variations in the dropping rate or size of the mercury drop, and the composition or viscosity of the test solution. That the step ratio is also independent of the temperature is indicated in Table I, and moreover the ratios are not affected by recorder sensitivities. Only two curves need be developed to fit any condition encountered in practice (Figures 1 and 2). They were obtained by polarizing 2-ml. portions of solutions containing known amounts of lead and cadmium. The total volumes and types of solutions used, the amount of cadmium added, and the polarizing conditions have been indicated on the charts. The current-voltage curves from -0.3 to -0.9 volt were obtained with the Leeds & Korthrup Chemograph which employs a Micromax ink recorder instead of the usual photographic tracing of the galvanometer swing. TABLEI. EFFECTOF TEMPERATURE ON LEAD-CADMIUM STEP RATIO Temperature
c.
24 30 35 40
(Drop r a t e = 10 drops in 30 seconds) P b Step Cd Step P b / C d Ratio Mm. Mm. 16.8 31.8 0.528 19.2 36.3 0.529 20.1 38.2 0.526 22.2 42.7 0.520 Av. 0 . 5 2 6
Procedure PREPARATION OF SAMPLES. The biological material was ashed at 500” C. as described in previous papers (3, 4,9). The ashed materials were brought into solution by addition of nitric or hydrochloric acid and distilled water and the solutions were rinsed into glass-stoppered Pyrex cylinders or volumetric flasks, the volumes being adjusted with distilled water as dictated by experience. ELECTROLYTIC ISOLATIOS AND CONCENTRATION. An aliquot or the entire sample, consisting of 5 to 20 grams of blood or solid tissue, 100 t o 250 ml. of urine, 0.1 to 0.15 gram of the ash of feces, or 1/20 of a day’s mixed food, is placed in a 50-ml. Pyrex beaker and the lead is collected on a platinum gauze electrode, 9 mm. in diameter and 25 mm. high, by a method described elsewhere ( 2 ) . The electrode is washed with hydroquinone solution (0.1 gram in 100 ml. of distilled water, and made just alkaline with ammonium hydroxide immediately before use), is dried, and the lead is stripped from it into 2 ml. of stripping solution (10 per cent tartaric acid, 0.08 N nitric acid, 6 micrograms of cadmium per ml.) in the cell over the anode pool of mercury. This cell is so constructed that when the gauze cylinder is inserted it is completely covered by the solution in the cell. [In dealin with very minute amounts of lead (1 to 10 micrograms) it is fesirable t o strip the lead into 1 ml. of solution, and for this purpose a plati-
INDUSTRIAL AND ENGINEERING CHEMISTRY
584
num gauze electrode 6.5 to 7 mm. in diameter and only 15 mm. high is employed.] Nitrogen (freed from oxygen by bubbling through ammoniacal cuprous chloride solution and then through dilute sulfuric acid) is passed slowly through the solution for 5 minutes to remove dissolved oxygen, and the solution is polarized from -0.3 to -0.9 volt at a suitable recorder sensitivity. The “step ratio” (Pb/Cd) is then obtained and the quantity of lead (corrected for the volume of solution polarized) is read from Figure 1.
TABLE11. RECOVERIES OF LEADADDED TO SYNTHETIC URINE ASH
-
Lead Added Micrograms
0 2 16 42 80
A
I i
I 60
Vol. 13, No. 8
(Samples correspond t o 100 ml. of urine) Lead Found --Polarographic MethodElectrolytic isolation a n d Combined extrac- Dithizone method concentration tion and electrolysis (1) Micrograms Micrograms Micrograms 0.0 0.0 0.0 0.0 0.2 0.2 2.2 2.5 2.8 2.7 2.3 2.3 16.5 15.3 16.0 15.0 16.3 17.3 39.0 39.5 40.0 40.0 40.8 41.3 80.0 78.0 80.0 82.0 79.0 79.0
TABLE 111. COMPARATIVE RESULTSON COMMON BIOLOGICAL MATERIAL Material FecesQ
Lead b y Polarographic Method Electrolytic Combined exLead by isolation and traction and Dithisone concentration electrolysis Method (1) Mg. Mg. Mg. 0.55 0.56 0.58 0.29 0.29 0.30
-
0.21 0.44
0.20 0.39
0.22 0.43
0.22 0.41
Urine
0,035 0.170 0.086
0.036 0.160 0.082
0.038 0.170 0.087
0.048 0.170 0.087
Blood
OO8N ntricacid 12 micrograms Cd ) Recorder renslttvity In, 1/3, 1/4
I
IO
20
30
12E.4D
0.065 0.080
... ...
Mg./iOO
Q.
... o:oi1 0.060
Mg./100
Q.
0.070 0.095 0.071 0.064
Mg./100 g . 0.065 0.085 0.075
0.070
Twenty-four hour samples. b Mixed food samples duplicating t h a t eaten by experimental subject in 24 hours. a
’
40
Mcroqrams P b / Z m l
FIGURE 1.
Mn. 0.58 0.32
Foodb
.Wg./iOO g.
0.40
Lead by Spectrographic Method (4)
CURVE
In dealing with feces, individual or mixed foods, or other samples containing relatively large mounts of copper and iron, *electrolysisin the presence of ammonium citrate and potassium cyanide is employed for one hour (2), or a double electrolytic technique may be used, consisting of an initial half hour’s electrolysis in the presence of ammonium citrate followed by an hour’s electrolysis in the presence of potassium cyanide (2). DITHIZONEISOL.4TION AND ELECTROLYTIC CONCENTRATION. ,4s before, either a suitable aliquot or the entire sample is used and the lead is first removed by means of dithizone (0.03 gram in 1000 ml. of chloroform), preferably in the presence of citrate and cyanide (1). After the dithiaone extract has been washed with 50 ml. of distilled water, the lead is shaken into 20 to 40 ml. of dilute nitric acid (1 ml. of nitric acid, specific gravity 1.40, in 100 ml. of distilled water). If the color of the dithizone extract has indicated that the amount of lead is low (less than 40 micrograms), the acid extract is placed in a 50-ml. beaker, 2 ml. of deleaded ammonium citrate (40 grams of citric acid per 100 ml.) are added, the solution is made just alkaline to phenol red, and the lead is electrolyzed onto the 9 X 25 mm. gauze electrode for 30 minutes. After the electrode has been dried (washing of the $electrodeis not necessary), the procedure is the same as that described above. In case less than 10 micrograms of lead is expected or indicated by the dithizone extract, the 7 X 15 mm. electrode i. used, so as to permit stripping of the lead into 1 ml. of solution. When more than 40 micrograms is present, analysis may be accelerated by eliminating the electrolytic step. If the lead content is sufficiently high, the acid washing of the extract may be polarized immediately after the addition of the proper amount of cadmium to serve as the internal standard, or the lead may be concentrated in a smaller volume of test solution. In the latter case the acid extract is evaporated to small volume (3 to 5 ml.); the solution is made just acid to phenol red; 1 ml. of a cadmium solution (90 micrograms of cadmium) is added; and the volume tis made up to 10 ml. in a glars-stoppered Pyrex cylinder. Two milliliters of this solution are polarized at recorder sensitivities indicated on Figure 2.
Results In Table I1 are listed recoveries of lead added to portions of a synthetic urine ash solution, in which 5 ml. are equivalent, to the ash of 100 ml. of normal urine (3). For purposes of comparison, results obtained for the same samples by the photometric dithizone method (1) have also been listed in this table. Results obtained on a number of random samples by the polarographic, spectrographic, and dithizone methods are recorded in Table 111. Except in the case of the blood samples, sufficient material was available to provide suitable aliquots for each of the four methods. The tissues listed in Table 1V were from a person with a known lead exposure,
I
I
A N A L Y T I C A L EDITION
August 15, 1941
which, however, was unrelated to his death. The results by four types of procedures are given when the sample was large enough.
585 Recorder %itivi+y
-0.9-
._
-.1
,
= 113
Step-mti0(0)~#&=0.465
Discussion From the standpoint of accuracy and coni-enience, either polarographic method compares favorably with the dithizone and spectrographic methods and its use for routine analytical purposes is fully justified. Results tend to be slightly lower than with the other methods, but the difference is insignificant. For certain materials the electrolytic technique is more time-consuming than the combination of extraction and electrolysis, but the former is so simple and requires so little attention that it can be carried out simultaneously with other laboratory work. I n addition, its manipulative steps are so distinctly different that its use gives additional confirmation of results obtained by other methods ( I , $ , 49). The extraction-electrolysis modification removes practically all extraneous metals, is highly flexible, and lends itself well to a number of obvious variations which greatly enhance its practical value. Frequently enough material is available to permit the use FIGURE 3. CURVES FOR SOLUTIONS O F ASHEDMATERIAL of aliquots yielding lead in sufficient amounts A B C D. Equal aliquots of same urine to permit direct polarization of the acid washA : Elebtrode not washed. Base, tartaric acid-nitric acid B . Electrode washed with alkaline hydroquinone solution. Base, tartaric acid-nitric acid ings of the dithizone extracts. C. Same as B , except base potassium chloride-hydrochloric acid solution D. Electrode washed with hydroxylamine-hydrochloric acid solution. Base same as -4 Attempts to polarize solutions of ashed material directly did not prove entirely satisfactory. I n the case of urine samples satisfactory cathodic curves were obtained when the soluterial a t the interface, which spread rapidly through the solutions were made just acid to prevent the precipitation of tion as soon as nitrogen was passed through it. This precipiphosphates, but the dilution required to keep all salts in solutate developed with as little as 100 micrograms of F e + + + per tion reduced the sensitivity of the method to such an extent ml. of solution and increased in amount with the quantity of as to limit its practical value (c, Figure 3). In addition, the the iron. Spectrograms of test solutions containing this prepresence of other reducible metals is troublesome; traces of cipitate and of the clear solution after settling of the deposit lead can be determined only if the quantities of iron, copper, showed that the precipitate was due to some unidentified and bismuth in the solutions are definitely limited, while mercury compound, either occurring in combination with mere traces of stannous tin, trivalent cobalt, and thallous ferric ions or precipitated by them. ions interfere because of the practical coincidence, in neutral or In most cases, therefore, the advantage of isolating the acid solution, of their half-ware potentialswith that of lead(l1). lead electrolytically or by extraction with dithizone is obIn addition to the possible masking effects of the iron steps, vious, while the necessity for concentrating lead becomes apcathodic curves a t high recorder sensitivities for solutions conparent from consideration of comparative ultimate sensitivitaining ferric iron are very poor, usually because of a high ties. Practical considerations limit the volume of the test limiting current (d, Figure 3). Ferric iron in the test solution solution to 1 ml., in which as little as 1 microgram of lead can reacted in some manner with the mercury of the anode pool be detected by the polarograph. This is a disadvantage, as (possibly by oxidation of the mercury) to give a smudgy macompared with spectrographic and dithizone methods. (One microgram of lead in 10 ml. of solution can be estimated spectrographically by using only 0.2-ml. aliquots, 3, 4. The ultimate sensitivity of the dithizone method seems to be TABLEIV. COMPARATIVE RESULTS ON HUMAN TISSUESQ limited by the magnitude of the rack blank. I n one proLead b y Polarographic cedure the latter has been reduced to 0.1 microgram, 1, Method ElectroComthereby making it possible to estimate a fraction of a microLead by lytic iso- bined exBpectroLead b y lation and traction gram of lead in any volume of solution which can be handled graphic Dithicone concenand elecin the rack equipment.) This limitation in performance on Tisaue Weight Method (4) Method (1) tration trolysis Grams , M Q . per 100 oran8s the part of the polarograph is not of practical significance, 5 leen 28.4 1.05 1.1s 1.12 1.10 however, since very few occasions will arise when it will not 2eart 30.1 0.050 0.032 0.035 0.045 be possible to isolate a t least 1 microgram of lead. Suprarenal 6.0 0.24 0.20 Stomach 26.5 0.085 0:695 0:080 0.070 Two stripping media-a 10 per cent tartaric acid-0.08 N Lung 49.0 0.39 0.40 0.36 * 0.35 LMuscle 29.0 0.045 0.03 0.025 0.036 nitric acid solution and a 0.1 Y potassium chloride-0.1 N Thyroid 4.5 0.62 0.57 hydrochloric acid solution-were effective in dissolving lead Small intestine 18.5 0.070 01060 0.06 01065 Rib bone 6.5 16.86 17.7 17.1 18.2 from the electrodes, but the former is preferable, especially a In most cases aliquots employed represent very small quantities of mawhen only the electrolytic method of lead isolation and conterial, 80 t h a t differences between results were often no more t h a n l microcentration is used. In order to obtain satisfactory polarogram. grams by this method, it was found necessary to wash the elec-
586
INDUSTRIAL AND ENGINEERING CHEMISTRY
trodes before stripping the lead from them. A number of wash solutions were tried; water was found to cause a slight loss of lead, and weak solutions of sodium sulfite and sodium hyposulfite were not satisfactory. A dilute solution of hydroxylamine hydrochloride did not affect the quantitative recovery of lead and the polarograms were greatly improved, but slightly low results were always obtained because of an artificial lengthening of the cadmium step. The use of hydroquinone solution, however, eliminated this difficulty and satisfactory polarograms were consistently obtained when the electrodes thus washed were stripped in the tartaric acidnitric acid base. When the electrodes were stripped in the hydrochloric acid-potassium chloride base, occasional curves (such as C in Figure 3) were obtained which were too irregular to permit accurate measurement of the steps. Acid and alkaline potassium tartrate media were also tried but these did not prove satisfactory, because of troublesome precipitates, high limiting currents, or fusion of the lead and cadmium steps. The latter reason no doubt accounts for Forche’s failure to use the step-ratio procedure in his method for lead determination in blood, even though he first suggested its use for analytical purposes (6). A number of the effectsjust discussed are illustrated by the polarograms reproduced in the lower section of Figure 3. In addition at a and b, respectively, of the upper section of the figure! polarograms for equal amounts of lead added to the tartaric acid-nitric acid and the potassium chIoride-hydrochloric acid bases are reproduced. The method of slopes used to obtain the step ratios, as well as the shift of the half-wave potentials for the lead and cadmium steps due to the change in composition of the bases, have been indicated for these polarograms. The sensitivity is somewhat less in the tartaric acid-nitric base, but this is compensated for by the improvement in the curves, permitting more certain measurements of the steps. At c and d, respectively, are illustrated cathodic curves for the direct polarization of solutions of ashed urine and feces samples. The aliquots chosen were concentrated to 10 ml. after being made just acid to prevent the precipitation of phosphates, and although 8 and 12 micrograms of lead were present, respectively, neither c nor d shows the presence of lead. In the case of d, a high limiting current, as indicated by the shift of the curve to the right, is attributed to the presence of ferric ion, since the characteristic precipitate described above formed in the test solution. In practice the direct electrolytic isolation and concentration of lead from prepared samples of biological material has been found effective except in occasional samples which contained bismuth and large amounts of copper. In many cases these interferences may be overcome by using smaller aliquots or by diluting the solution to be polarized. If distinct steps are not obtained by this procedure, it is best to repeat the analysis on a fresh aliquot by the modified method. Bismuth, if present, can be extracted with dithizone a t pH 2 as described in previous papers ( I , 9). The possibility of isolating and concentrating bismuth electrolytically suggests that a similar method might be developed for its polarographic determination. I n this case, as the half-wave potential of bismuth is considerably below that of lead, relatively large amounts of the latter should not interfere.
Vol. 13, No. 8
An adaptation from spectrographic analysis of the internal standard principal permits the derivation of stable working curves, which are not affected by variations in temperature, drop size or rate, viscosity or composition of the solution to be polarized, or by changes in the recorder sensitivity used. The accuracy and practical sensitivity of the method approach those of the spectrographic and dithizone methods.
Literature Cited (1) Bambach, K., IND.ENO.CHEM.,Anal. Ed., 11, 400 (1939). (2) Bambach, K.,and Cholak, J., Ibid., 13,504 (1941). (3) Cholak, J., Ibid., 7,287 (1935). (4) Cholak, J., and Story, R. V., Ibid., 10, 619 (1938). (5) Forche, H. E., Mikrochemie, 25, 217 (1938). (6) Forche, H. E., “Polarographische Studien”, dissertation, Leipzig, 1938. (7) Hamamoto, Orient. J . Dis. Infants, 19, 18 (1936). (8) Hohn, H., “Chemische Analysen mit dem Polarographen”, Berlin, Julius Springer, 1937. (9) Hubbard, D.M.,IND.ENO.CHEM.,Anal. Ed., 9, 493 (1937). (10) Kolthoff, I. M.,and Lingane, J. J., Chem. Revs., 24,1 (1939). (11) Perley, G.A,, Trans. Electrochem. Soc., 76,91,1939. (12) Shikata, M.,Tachi, I., and Hozaki, N., Bd1. Agr. Chem. SOC. Japan, 3,52 (1927). (13) Teisinger, J., 2.gw. ezptl. Med., 98, 5 Heft, 520 (1936). (14) Walkleu, A.. Australian Chem. Imt. J . & Proc., 5. 291 (1938). (15) Yoshida, S.,Orient. J . Dis. Infants, 23, 15 (1938).
Volumetric Tubes for Small Volumes FREDERIC E. HOLMES The Children’s Hospital Research Foundation and Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio
H E appearance of the description of a volumetric flask T b y caley ( I ) prompts the publication of other modifications which have been used in this laboratory. E: Figure 1, A , shows a form superficially similar to the old Folin sugar tube. The contents of the bulb are mixed in the same manner as in Caley’s flask, except that the tube cannot be brought quite to the horizontal position; some solution is -
U 50mm.
10 ml.
5 ml.
Summary A polarographic method is described for the estimation of lead in biological material, based on electrolytic isolation and concentration of lead followed by stripping into very small volumes of solution. Two methods of concentration are presented: (1) a purely electrolytic technique, and (2) an initial dithizone isolation, followed by electrolytic concentration when very small amounts of lead are present.
A FIGURE 1. VOLUMETRIC TUBES
B
