504
INDUSTRIAL AND ENGINEERING CHEMISTRY
The pressure was measured to 0.1 mm. of mercury on a Utube mercury manometer, with a calibrated, glass, silverbacked scale. The liquid filled the micropycnometer to a height of about 25 111111. and this could be estimated to about 0.2 mm. The diameter of the capillary was about 1 mm. cc. per mm. of length. and its cross section about 8.5 X Thus the calculated error on the measurements in Table I is about 0.5 to 1 per cent. This accuracy can readily be improved upon by using a smaller capillary and refining the other measurements-e. g., using a smaller system and n-eighing on a microbalance to 0.01 mg.
Vapor Pressures The vapor pressures have been plotted in Figure 2 and the experimental points are recorded in Table 11. Over the range of temperatures measured they fit I'ery 11-ell the Clausius-Clapeyron equation : log P = - A / T
+B
when P is in mm. of mercury at 25" C. and T i q in degrees Absolute. From the slope of the straight lines we can calculate the heats of vaporization in this range of temperature (Table 111). I n these measurements the pressures were read to 0.1 mm. of mercury and the temperatures to 0.5" C. Care was taken to ensure the establishment of equilibrium which was approached from both higher and lower temperatures. The calculated error in the constants recorded in Table I11 is about 0.5 to 1 per cent.
TABLE 111.
HEATS O F
Cornp o u n d Cyclopentane 2-Pentene 1-Pentene
Vol. 13, No. 7
V.4PORIZATION .4ND CLbUSIUS-CLAPEYROK CONSTANTS A 1646.0 1512.5 1259.5
B
S 0880
7 7945 6 9570
H"**. Kcal./.mole 7.524 6 913 5.757
The measurements of vapor pressures in the case of small samples have necessarily been limited to a narrow range of low pressures, because of the large size of the system. This limits the use of these vapor pressures in the determination of unknowns, since small amounts of volatile impurities introduce a rather sizable error in this range. Furthermore, the pressures are small and therefore not quite so accurate. -4n apparatus has recently been devised to measure vapor pressures on a micro scale up to the boiling point where small amounts of impurities do not create great errors and where the mole per cent can be determined. This will be fully described in a later paper.
Literature Cited Blacet and Leighton, IND. ENG.CHEM.,Anal. Ed., 3, 276 (1931). Blacet and MacDonald,Ibid., 6, 334 (1934). Blacet, MacDonald, and Leighton, Ibid., 5 , 272 (1933). Blacet, Sellers, and Blaedel, Ibid., 12, 356 (1940). Blacet and Volman, Ibid., 9, 44 (1937). Dolliver, Gresham, Kistiakowsky, and Vaughan, J . Am Chem SOC.,59, 831 (1937). (7) Hurd, Goodyear, and Goldsby, I b i d . , 58, 235 (1936). (8) Kistiakowsky, Ruhoff, Smith, and Vaughan, Ibid.. 58, 137 (1936).
(1) (2) (3) (4) (5) (6)
Electrolytic Deposition of Lead from Biological Material 6.4RL BAMBACH AND JACOB CHOLAB Kettering Laboratory- of -4pplied Physiology, University of Cincinnati, Cincinnati, Ohio
I
S AN attempt t o develop a polarographic method for the determination of microquantities of lead in biological material, it was found that the necessary separation and concentration of the metal could be accomplished electrolytically, provided certain requirements were fulfilled. I n most of the methods reported in the literature for this separation (3,4, 9, IO, 13, 14, 15), lead is first precipitated as the sulfide or oxalate, in order to remove substances which p-ould otherwise interfere with the electrolysis. Any procedure involving precipitation and filtration introduces opportunities for both loss and contamination when microquantities of lead are being handled and therefore a method was sought for the direct electrolysis of the prepared sample. Such a procedure would be even simpler than the one recommended by the Association of Official Agricultural Chemists ( I ) , in which the lead is electrolyzed following its extraction with dithizone. A few methods have been reported in which the lead in biological material is separated as lead dioxide by direct electrolysis of the prepared sample (5, 8, 12) or as metallic lead b y electrolysis of fresh urine ( 7 ) , but the authors were not able to secure quantitative separations by these procedures. It is possible that failure to obtain a complete recovery of trace8 of lead escaped notice at the time the meth-
ods were devised because precise microanalytical procedures for lead estimation were not then available. These low recoveries are due a t least partially to the fact that the electrolyte must be kept acid (pH 3 to 4) in order to prevent' precipitation of calcium and lead phosphates, and also to the interference caused in the quantitative deposition of lead by phosphate and iron, which are always present in biological material (illustrated in Table I). I n all experimental data reported in Tables I and 11, the quantity of lead deposited on the electrode was determined in the following way: The lead was stripped from the electrode with about 10 ml. of dilute nitric acid (10 ml. of nitric acid, sp. gr. 1.40, per liter), transferred to a graduated separatory funnel, and 1 ml. of deleaded hydroxylamine hydrochloride solution (20 grams per 100 ml., 2 ) was added. The solution was brought to a volume of 50 ml. with the dilute nitric acid and redistilled ammonium hydroxide was added until a pH of 2 was reached (orange color of rn-cresol purple); from this point the procedure was the same as that previously described (2) beginning xith "Final Estimation of Lead".
Procedure The sample is prepared by destroying the organic material at 500' C . as described in previous papers (6, 11). The ash is dis-
solved in nitric and hydrochloric acids and double-distilled water.
July 15, 1941
ANALYTICAL EDITION TABLEI. DEPOSITION OF LEAD
P b Present
Lead On anode, electrol zed by A. A. C. method
Micrograms
Micrograms
50
46
50
24
50
..
19.3
50
17
..
..
49.0
7 -
Sample Pb(NOa)z solution Pb(N0a)r solution 0.1 gram of NaHtPO4 Same as above 3 ml. of ammonium citrate solution Pb(N0a)z solution 5 mg. of Fe as FeCls Same as above 3 ml. of ammonium citrate solution Food 2786 Same as above 5 ml. of ammonium citrate solution
+
+
+
+
+
50 22, determined by photometrir dithizone method
8.
Sone
Found On cathode, electrolyzed by new method (single) Micrograms
..
..
22.0
(2)
iln aliquot or the entire sample, representing 10 to 25 grams of blood, 100 to 250 ml. of urine, 0.1 to 0.15 gram of fecal ash, or 1/20 of a day's mixed food, is placed in a 20- to 100-ml. beaker. If the lead on the electrode is to be determined by dithizone, it is not necessary to prevent the deposition of iron, copper, or other substances which might interfere with the determination by other methods. Five to 10 ml. of deleaded ammonium citrate solution (40 grams of citric acid per 100 ml., 2 ) are added to the sample, and the mixture is made alkaline to phenol red with distilled ammonium hydroxide, and diluted with 2 to 5 times its volume of double-distilled water. An electrolytic apparatus similar to that described by the Association of Official Agricultural Chemists ( 1 ) is employed and the solution is electrolyzed at 5 to 6 volts for 30 to 60 minutes, the cathode serving as the rotating electrode. With the authors' equipment, the current varies from 200 to 500 milliamperes. The lead can then be stripped from the cathode with dilute acid and determined as indicated above. The results of analyses by this method, compared with those obtained by dithizone ( 2 ) and spectrographic (6) procedures, are given in Table 11. If necessary, the electrodes can be washed with 100 ml. of deleaded hydroxylamine hydrochloride solution (0.2 gram per 100 ml.) or 100 ml. of hydroquinone solution (0.1 gram per 100 ml.). This washing should be done by siphoning, so that the electrodes are not exposed to the air at any time, and the electric current should not be shut off while the electrodes are in the solution. Instead of washing the electrodes, a method of double electrolysis can be employed in which the sample solution is electrolyzed as usual, the cathode is stripped in dilute nitric or hydrochloric acid, 1 ml. of the ammonium citrate solution previously mentioned is added, and the solution (made alkaline to phenol red) is again electrolyzed. I n certain samples, such as those of feces and mixed foods, the copper and iron content is so high as to interfere with subsequent determination of the lead on the electrode by the polarograph. Deposition of those metals, particularly iron, can be prevented largely by the addition of 2 ml. of deleaded potassium cyanide solution (10 grams per 100 ml., 2 ) to the solution before electrolysis. This delays the plating action, however, and it is then necessary to electrolyze for 1 to 2 hours. If desired, greater purity of the lead deposit may be secured by double electrolysis with potassium cyanide in each solution, although the deposit is never completely free from certain other metals.
Possible Applications The method of direct electrolysis, combined with a photometric dithizone procedure for determination of the lead on the electrode similar to t h a t referred to above, provides a rapid and precise method for the determination of lead in biological material. A few modifications of the dithizone procedure would be necessary to make it applicable to routine work. For instance, instead of three standard dithizone solutions, each suited to a different range of lead concentrations, one solution only could be employed, and varying quantities used according to the amount of lead present. The strength of the solution could be so adjusted that, 5 ml. would be equivalent to 10 micro-
SO5
grams of lead; if a 5-ml. portion of the solution turned red after the lead-containing solution was shaken with it, additional quantities of the standard could be used, in 10-ml. portions, until an excess of dithisone was present; the dithisone solution would not be drained out between successive additions, and the color of the final mixture could then be read photometrically. While such a method would not be more accurate than the better dithizone extraction procedures, it would probably be much more rapid, provided a number of electrolytic units were available and could be used simultaneously.
A polarographic method of determining the lead deposited on the cathode will be described in a later paper (sa). It is possible that other ways of determining the lead on the electrode, such as titration or colorimetric methods, can also be worked out, particularly in view of the fact that the lead deposit is often very pure. Summary In a new method for the electrolytic deposition of traces of lead from biological material, the solution of the ashed sample is electrolyzed directly, without previous separation of the lead. The electrodes can be washed and other metals can be complexed, so that the lead deposit on the cathode is often almost entirely free from other substances. The lead can then be determined b y the use of dithizone, the polarograph, or by other methods.
----
T.~BI.E 11. DEPOSITIOS OF LEAD Sample
Sample Electrolyzed
Microarams Pb(NOd2 P b (Nos)2 Crine 2919 Urine 2919 Urine 3948 Blood 4397 Blood 4011 Blood 3687
Lead Found-By dithizone extrac- B y spectroB y electrolysis tion ( 8 ) graph ( 6 )
.Microoranis 9.60 9.Sb
10 10
92 9.2 66 Grants 8 8 5 17 5 0.1-0.15ash 0.1-0.15 ash 0.1-O.15ash 0.1-0.15 ash 0 1-0.15 ash 1/50 day's food
.Me /loo g 0 067" 0 OB6b 0 046
0.29~ 0.70;
0.71 0.31; 0.31 0.022 .lfQ./lOO
.. ..
0 077
0.077 0.087 0.160"
-MQ Feces 3553 Feces 3499 Feces 3499 Feces 3226 Feces 3226 Food 0786
Ma. 11.
0.17 M Q /IO0 Y. 0 07 0 055
.If y 0.30 0.71 0.'315 0 .'dz2
8,
.Ma./l.
.. ..
.. 0 .'i7 .Mg./lOO Q. 0 065 0.06 0 045 MQ.
0.285 0,696
..
... . .. .Mg./lOO
g.
Tissue (suprarenal) 3894 10 0.057 .. 0.045 a Electrodes washed with hydroxylamine HCI solution. b Double electrolysis, no K C N present. C K C N present, electrodes washed,with hydroxylamine HCII solution. d KCN present, double electrolysis. e Single electrolysis, K C N present.
Literature Cited (1) Assoc. Officialdgr. Chem., "Official and Tentative Methods", 4th ed., pp. 381-3 (1935). (2) Bambach, Karl, IND. ENG.CHEM.,Anal. Ed., 11, 400 (1939). (3) Barth, E., Virchow's Arch. path. Anat., 281, 146 (1931). (4) Beck, H., and Straube, G., Klin. Wochschr., 18, 242, 356 (1939). (5) Berg, Ragnar, Biochem. Z., 198, 420 (1928).
(5a) Cholak, J., and Bambach, Karl, IND. ENG.CHEM.,Anal. Ed., 13, in press (1941). (6) Cholak, J., and Story, R. V., Ibid., 10, 619 (1938). (7) Cooksey, T., and Walton, S. G., Analyst, 54, 97 (1929). (8) Danckwortt, P. W., and Jurgens, E., Arch. Pharm., 266, 367
(1928). (9) Denis, W., and Minot, A. S., J. Biol. Chem., 38, 449 (1919). (10) Francis, -4. G., Harvey, C. O., and Buchan, J. L., Analyst, 54, 725 (1929). (11) Hubbard, D. M., IND.ENG.CHEM., Anal. Ed., 9, 493 (1937). (12) Lehmann, V., Z. physik. Chem., 6 , 1 (1882). (13) Miiller, H., Z. anal. Chem., 113, 161 (1938). (14) Necke, A, and Muller, H., Angew. Chem., 48, 259 (1935). (15) Schmidt, P., Weyrauch, F., Necke. A., and Muller, H., 2.gss. e s p t l . M e d . , 94, 1 (1934).
