Determination of Lead by Dithizone Modifications and Improvements of the Hubbard-Clifford-Wichmann Method as Applied to Biological Material KARL BAMBACH Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio
D
ITHIZOKE has been used in this laboratory for the past three years as a reagent for the determination of lead in biological material, and the experience gained in about ten thousand lead analyses has led t o certain modifications and improvements in the method reported by Hubbard (Y), who based his photometric dithiaone procedure upon work done by Clifford and Wichmann (6) and by WBkins, Willoughby, Winter, and their co-workers (IO, 11, 1.3). The improvements described in this paper have made possible increased accuracy and a saving of time, and have resulted in keeping the rack blank down to the very low figure of 0.1 microgram.
Apparatus The apparatus employed is essentially the same as that described by Hubbard ( 7 ) . The performance of the neutral wedge photometer, however, has been improved by a few changes. A small compensating wedge of Bausch & Lomb neutral “C” glass has been placed in the light path, as suggested by Clifford (6),in order to make the comparison fields of the instrument “shade off” similarly. I n addition, the compound gelatin filter has been replaced by a permanent glass filter with maximum transmission a t 510 millimicrons. These changes necessitated a stronger light source and accordingly 50-candlepower bulbs have been substituted for the 32-candlepower bulbs originally used. A very convenient device, suggested by R. R. McNary, of this laboratory, consists of special rotary funnel racks (Figure 1)fitted with opaque glass bases. The metal rings which hold the funnels have been rubber-plated b y the Anode process. This rubber covering resists the reagents used in the analysis much better than any type of paint available. Glassware and other equipment used are similar to that described by Hubbard ( 7 ) .
A substantially saturated solution containing 50 grams of potassium cyanide in sufficient water to make 100 ml. is repeatedly shaken with portions of dithizone in chloroform (30 mg. per liter) until the lead is removed. Part of the dithiaone dissolves in the aqueous phase but sufficient remains in the chloroform to color it and to indicate when the lead has been completely extracted. Most of the dithizone in the aqueous phase can be removed, if desired, by repeated extractions with pure chloroform. The strong potassium cyanide solution is then diluted with redistilled water to the proper strength (10 grams per 100 ml.). It is not necessary to Alter the solution. (If instead of the concentrated solution, the final one is shaken with dithizone in chloroform in an attempt to delead it, the increased alkalinity of this dilute solution causes the removal of all the excess dithizone from the chloroform and renders the complete extraction of the lead more difficult.)
Hydroxylamine hydrochloride solution (20 grams per 100 ml.) is made substantially lead-free as follows: Twenty grams of hydroxylamine hydrochloride are dissolved in sufficient water t o make about 65 ml. and a few drops of mcresol purple indicator solution are added. Concentrated ammonium hydroxide is next added until a yellow color results. Sodium diethyldithiocarbamate in water (an approximately 4 per cent solution) is added in sufficient quantity to combine with all the lead (and most other metals) present and to leave a considerable excess. After a few minutes the organo-metallic complexes and the excess reagent are completely extracted with chloroform. The absence of a yellow color in the chloroform when a portion of the chloroform extract is shaken with a dilute solution of a copper salt indicates when this point is reached. Redistilled hydrochloric acid is then added to the hydroxylamine hydrochloride solution until the indicator turns pink, and redistilled water is added to make the final volume 100 ml. It is not necessary to filter the solution. Other reagents are purified as described by Hubbard (7). By using these carefully purified reagents and avoiding other sources of contamination (such as removing dust in the room by air filtration) it has been possible to show a rack blank of not more than 0.1 microgram and to keep this blank consistently low from day to day. Obviously, a consistently low blank increases the significance of analytical results when the sample contains only a small quantity of lead (below 10 micrograms), Since fecal samples are prepared for analysis by dry ashing, the rack blank of 0.1 microgram represents the total blank for such analyses, and if redistilled acids are used for the preparation of other types of samples, the total blank
Reagents Ammonium citrate solution (40 grams of citric acid per 100 ml.) is deleaded by shaking it with dithiaone in chloroform after sufficient ammonium hydroxide has been added to make the solution alkaline to pbenol red. Potassium cyanide solution is rendered lead-free by the following procedure : 400
JULY 15, 1939
ANALYTICAL EDITION
may also remain in the order of 0.1 microgram. However, in practical routine work where large quantities of acid are used to prepare large samples of urine and mixed foods, the reagent acids used are restricted to uniform lots which are analyzed periodically, and a small blank results from the acid used in the preparation of the sample. The author believes that the blank of 0.1 microgram is the lowest consistent figure so far reported for this type of lead analysis.
Experimental Clifford and Wichmann’s method (6)is not directly applicable to the analysis of certain biological material containing bismuth or large amounts of inorganic salts, such as feces and mixed foods. Inorganic salts often interfere by oxidizing the dithizone in the initial extraction, thus preventing the choice of the proper standard dithizone solution to be used in the pJRotometric step. I n addition, small amounts of salts may be entrained by the dithizone-chloroform solution in the first extraction and cause interference in the latter part of the analysis. Because of these factors Hubbard (7) found it necessary to use two extractions in isolating the lead and choosing the proper standard dithizone solution. He also tested each sample for bismuth and removed it if present. It has been reported (1) that the oxidation of dithizone in the first extraction referred to above could be eliminated by the use of hydroxylamine hydrochloride. This would make possible a shortened procedure, in which the lead could be estimated roughly in the first extraction. However, it was felt that hydroxylamine might reduce the tin which is always present in feces and mixed foods so that the characteristic interference due to stannous tin would result. [As stated by Hubbard (7) and Laug (Q),this interference does not normally
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occur when biological samples are prepared by the usual oxidizing methods.] I n order to determine whether hydroxylamine would cause interference when used in analyses of this type in the presence of tin, prepared samples of feces and mixed foods were analyzed by Hubbard’s method with and without the addition of hydroxylamine, and were also analyzed for tin and lead by the spectrographic method used in this laboratory ( 3 ) . An inspection of Table I shows that hydroxylamine did not affect the results, although the spectrograph showed the presence of relatively large quantities of tin. TABLEI. EFFECTOF HYDROXYLAMINE Sample
Lead (Hubbard’s Methodla Without hyWith hydroxylamine droxylamine
Mu.
MQ.
Food, 1886 0.16 0.16 Food, 2198 0.34 0.34 Feces. 2243 0.56 0.55 a Alisuots of one tenth of t h e sample were used.
Spectrographic Method Tin Mu. Mu. 0.16 3.75 0.35 7.8 0.56 3.0
Lead
Salts entrained by the chloroform in the first extraction often make uncertain the bismuth test as described by Hubbard. This has been recognized by Hubbard in his work on a dithizone method for the determination of bismuth (8). These interfering salts, however, can be removed by washing the dithizone-chloroform extract with water; if this is done, tests have shown that as little as 3 micrograms of bismuth can then be detected by the routine test. Since no citrate is used in the second extraction step in Hubbard’s method, occasional samples high in both lead and phosphate may present difficulties due to the fact that phosphate entrained by the chloroform and carried over to the aqueous phase of the second extraction may cause the precipitation of lead phosphate. The loss will not take place if sufficient dithizone solution to extract most of the lead is added immediately after the nitric acid solution is made alkaline. I n the modified method here presented this precipitation of lead phosphate cannot occur. The loss of lead which takes place when the mixed color is not developed immediately after the addition of the ammonia-cyanide mixture has been attributed by Clifford (4) to the presence of phosphate as a n impurity in the potassium cyanide used. It is very likely that the phosphate causing the interference is often due not only to the potassium cyanide but also to entrainment by the chloroform in the first extraction. So far thallium has not been encountered in samples of biological material and no attempt has been made to include a test for its presence in the routine method. I n many analyses the absence of thallium has been confirmed by spectrographic observations.
Modified Method PREPARATION OF SAMPLES. Samples are treated as previously indicated (3, 7). ISOLATION OF LEAD. The procedure is similar to Clifford and Wichmann’s and to Hubbard’s but manipulative changes have been introduced and the method has been shortened. The aliquot of the prepared sample is treated as described by Hubbard, except that 1 ml. of
FIGURE 1. ROTARY FUNNEL RACKS
deleaded hydroxylamine hydrochloride solution is added to each sample after the addition of
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the ammonium citrate. The lead extraction is started with 5 ml. of dithizone solution and the color is noted in order that the proper standard dithizone solution may be chosen in the final lead estimation. (The lead usually is not extracted quantitatively with each portion of dithizone solution, because of the various salts present. Instead of 50 micrograms of lead, usually only 40 micrograms will be extracted by each 5 ml. However, with practice the quantity of lead actually present can be estimated from the color of the dithizone solution with surprising accuracy. When the quantity of lead present is less than 10 micrograms this is recognized by the distinctive greenish blue color of the 5-ml. portion of dithizone solution.) TABLE 11. CHECKANALYSES Lead Added Micrograms 0
5 25 50 75 100
Lead Found Micrograms
