Microdetermination of Lead bv Dithizone J
With an Improved Lead-Bismuth Separation KARL BAMBACH AND ROLAND E. BURKEY Kettering Laboratory of Applied Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio
D
ESCRIPTIOKS of the dithizone method for lead determination used in this laboratory were published in 1937 (6) and again in 1939 (1) but since that time several modifications have been introduced into the procedure. Of particular importance, the bismuth test has been replaced b y a lead-bismuth separation which results in a significant saving of time; this is given here in its entirety. At present, since details of the method as applied to various biological materials have been published in separate articles, it is necessary to consult a t least four papers in order to obtain the complete procedure (1-6).
tractions with dithizone will remove the metal completely. Bismuth is not a common constituent of biological material, yet each sample for lead determination must go through t b rather tedious bismuth test in order to make the analysis specific. TABLEI. EFFECTOF pH
ON
LEAD-BISMUTH SEPARATION
(Feces 293)
Old method ( 1 ) pH 3.4 no bismuth added DH 3.4: 100 micrograms of bismuth added PH 3 . 6 no bismufh added U H 3 . 6 : 100 microerams of bismuth added PH 4 . 0 ' no bismutTh added p H 4 . 0 : 100 micrograms of bismuth added
Standard dithizone solutions can be kept for months without detectable decomposition if certain precautions are observed. They should be stored in the dark, in a refrigerated space, and in glass-stoppered Pyrex containers. Whenever the chloroform used anywhere in the analysis is shaken with water, alcohol (about 0.5 per cent by volume is sufficient) should be added to it after the water is separated. Thus alcohol should be added after the chloroform is shaken with hydroxylamine solution in preparing a dithizone solution; it should be present in the bottle in which chloroform to be reclaimed is collected; it should be added to the chloroform after it is shaken with sulfuric acid in the reclaiming procedure; and the purified chloroform should be distilled into a receiver containing alcohol. The purified dithizone supplied by the Eastman Kodak Company is satisfactory for all dithizone solutions; special treatment is not necessary.
Lead Found Micrograms 57, 58 57 57 54 55 50 51
If, instead of using dilute acid-for example, 1 per cent nitric acid-to strip the metals from the dithizone solution, one uses an aqueous solution set a t p H 3.4, the lead and bismuth are separated and no subsequent bismuth test or extraction is necessary. When relatively large quantities of bismuth are present a slight amount is carried over, but this can be completely extracted by the addition of one portion of dithizone solution. Elimination of the bismuth test by this procedure results in saving about 25 per cent of the working time required by the dithizone method previously described, so that a single analysis on a prepared sample can be carried out in less than 20 minutes. With 3 hours' help on the part of a technician, between 50 and 60 prepared samples can now be handled by the analyst in 7 hours. The p H in this separation must not vary widely from 3.4, and in order to keep entrained salts from changing this factor it was found necessary to use a dilute Clark and Lubs buffer (potassium acid phthalate-hydrochloric acid). Also, in order to have the correct buffer system in operation in the final step when the photometric estimation of lead is carried out, the proper concentration of ammonium nitrate had to be present. This was accomplished by using 2 per cent nitric acid, setting the p H a t 3.4 with ammonium hydroxide, adding the proper quantity of buffer, and then diluting the solution with water to twice the volume of nitric acid used. The pH of the solution is very important; if the solution is too acid some bismuth enters the aqueous phase, causing a n apparently high lead recovery, while if it is too alkaline some lead will
The quantity of dithizone in the standard solutions used in the final step has been increased by 25 per cent. It was found that this eliminated a tendency toward deviation of the calibration points from a straight line when the upper limits of capacity of the solutions were reached. Although this method of lead determination was devised primarily for the analysis of biological material, it is capable of wide application. Lead in water, toothpaste, and most chemicals can be determined with little or no modification of the procedure.
The Lead-Bismuth Separation I n the various dithizone methods for the determination of lead in biological material (1,6,8, IO),the lead (and bismuth, if present) is extracted from the sample solution by shaking with dithizone in chloroform or carbon tetrachloride, the metals are stripped from the dithizone by shaking with dilute acid, and a test for bismuth is then made. If bismuth is found it is usually removed by extracting the aqueous solution with dithizone a t p H 2 to 3. At this p H no lead enters the dithizone phase, but bismuth is slowly extracted, so that repeated ex-
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November 15, 1942
ANALYTICAL EDITION
TABLE 11. SYNTHETIC SAMPLES CONTAINING LEADAND BISMUTH
Sample
A B C D
Lead Added Micrograms 0 (Blank o n synthetic urine ash) 2.5
NO bismuth added Micrograms 0.3
Lead Found 10 micrograms of bismuth added Micrograms 0.3
100
micrograms of bismuth added Micrograms 0.2
24
I D
TABLE 111. SAMPLES CONTAINING BISMUTH (Xcrograms of lead found) No Bismuth Added Bismuth Added Old New 10 100 method (1) method micrograms micrograms Urine Urine Feces Food Food
343 378 386 920 1191
4.6 14.5 80 14 37
4.6 14 79 14 38
4.6 14 80 14 37.5
4.6 14 80 14 37.5
stay in the dithizone-chloroform phase (cf. Table I). Considering the difference in the types of solutions analyzed, these results agree fairly well with those reported by Clifford and Wichmann (4) and with the rough chart proposed b y Wichmann (9). After this lead-bismuth separation had been used for some time a somewhat similar one was described b y Kluchesky, Longley, and Kozelka in a dithizone method for the determination of bismuth (7). However, these workers did not use a buffer, which would make their method inapplicable to the analysis of samples of larger volume, especially where large quantities of dithizone solution must be used for extraction such as are commonly*employed in the determination of lead. In order to show the reliability of the method and the completeness of the lead-bismuth separation, the results obtained on samples with known quantities of lead added to deleaded salts simulating the ash of normal urine (2),some of them contaminated with bismuth, are shown in Table 11. Table 111 shows the results obtained b y the new method on samples of urine, feces, and mixed foods which had also been contaminated with bismuth. A further comparison of results b y the old and the new method is included in Table IV. The data in these tables indicate that the simplification and shortening of the old method has been accomplished with no loss of accuracy; quantities of lead below 10 micrograms can be determined with an error of less than 1 microgram, while the error with quantities greater than 10 micrograms is about 3 to 6 per cent. When the quantity of bismuth in the sample exceeds 10 micrograms (approximately) it is advisable to shake the aqueous extract containing the lead at p H 3.4 with one 5-ml. portion of dithizone solution. The presence of this much bismuth may be recognized easily b y the fact that the dithizone extract does not return to its original green color when shaken with the p H 3.4 buffer solution; the practised eye perceives the off-color readily. By using the additional extraction, the last trace of bismuth is removed from the aqueous phase. Lead determination in samples containing relatively large quantities of bismuth may be facilitated b y making use of the fact that in the original dithizone extraction the lead is completely removed long before the bismuth, and that, therefore, when the color of the initial portion of dithizone extract shows the presence of both lead and bismuth, extraction need be continued only until it is apparent that most-not all-of the bismuth has been removed from the sample solution. A t this point one can be sure that all of the lead has been extracted
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and the sample can be discarded before the color of the last portions of dithizone extract remains a n unchanged green.
Complete Procedure for Lead Determination O F SAMPLES. Urine. Concentrated nitric acid (10 per cent of the sample volume) is added to the measured sample; it is evaporated to dryness on a hot plate or steam bath, and ignited at 500" C. in a silica or Pyrex evaporating dish. The ash is moistened with nitric acid, dried, and ignited for a few i n utes until it is white; then it is dissolved in dilute nitric acid and is ready for analysis. Filtration is not necessary. Feces. The sample is dried on a hot plate or steam bath to constant weight in a tared silica or Pyrex dish, placed on an electric heater a t a low red heat until all volatile matter which would otherwise flame in the muffle furnace has been driven off, after which it is ignited at 500" C. The ash is weighed, dissolved in a mixture of hydrochloric and nitric acids, and is ready for analysis. Filtration is not necessary and should not be employed, since the removal of silica in appreciable quantities will result in the loss of entrained lead unless the preci itate is very thoroughly washed. Foods. The weighed sampre of mixed food is digested with concentrated nitric acid on a hot plate or steam bath until all lumps have disappeared. This may require more than a week, with repeated additions of acid. If a food homogenizer, such as a Waring Blendor, is used the time may be materially shortened, especially since aliquants may then be employed. (Unless samples can be made homogeneous by this or some similar method, the entire sample must be analyzed.) The sample is evaporated to a sirupy consistency, transferred to a silica or Pyrex evaporating dish, and concentrated sulfuric acid is added (15 ml. for an average day's mixed food). The dish is then placed on an electric heater a t a low red heat and treated as described under "Feces". Tissues. In general, the weighed sample is treated in the same way as a sample of mixed food. Blood. The sample is transferred to a tared silica or Pyrex evaporating dish and weighed, after which concentrated nitric acid is added (5 to 10 ml. per 10 rams of blood). The mixture is evaporated to dryness on a hot $ate or steam bath, placed on an electric heater a t a low red heat, and i nited in a muffle furnace at 500' C. A few milliliters of hydrochyoric acid are added to the ash, then nitric acid, and the dish is heated on the hot plate until the sample is dissolved. It is then ready for analysis. Sote. All glassware should be cleaned with a sulfuric acidchromic acid mixture and with dilute nitric acid (1 1). It is necessary to avoid even slight contamination of samples with dust, especially during the evaporations. If reagent acids are used in the preparation of samples, lead determinations should be made on them for deduction as reagent blanks. This can be avoided by using distilled acids. REAGESTSAXD APPARATUS.Ammonium Citrate Solution. Four hundred grams of citric acid are dissolved in water and
PREPARATION
+
TABLE IV. COMPARISON OF RESULTS BY Two METHODS Samule Urine
956 960 962 964 978 992 996 998 1008
Lead Found Old New method (0 method Microoroms Microgram8 23 23.5 4.1 3.8 5.5 4.5 6.8 6.5 21 20 29.5 29.5 11.5 11 7.5 7.5 3.2 2.9
These samplea contained bismuth when received; over 200 micrograms were present. (1
i
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INDUSTRIAL AND ENGINEERING CHEMISTRY
sufficient reagent ammonium hydroxide is added to make the solution alkaline to phenol red. The solution is diluted to 1 liter with water and purified by shaking it with repeated portions of a solution of dithiaone in chloroform until the dithizone retains its original green color. Distilled Ammonium Hydroxzde. Reagent ammonium hydroxide is distilled into double-distilled water which is chilled in an ice bath. Hydroxylamine Hydrochloride Solution. Twenty grams of hydroxylamine hydrochloride are dissolved in sufficient water to make about 65 ml. and a few drops of m-cresol 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 cop er salt indicates when this point is reached. Distilled hydrochtric acid is then added to the hydroxylamine hydrochloride solution until the indicator turns pink, and double-distilled water is added to make the final volume 100 ml. It is not necessary to flter the solution. Potassium Cyanide Solution. A practically 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 dithizone dissolves in the aqueous phase but enough 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 double-distilled water to the proper strength (10 rams per 100 ml.). It is not necessary to filter the solution. If instead of the concentrated solution, the final one is shaken with dithiaone 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,) Dithizone Extraction Solution. One liter of chloroform is shaken with 100 ml. of water containing about 0.5 gram of hydroxylamine hydrochloride, which has been made alkaline to phenol red with ammonium hydroxide. The chloroform is drained off, and 30 mg. of dithizone are dissolved in it. Approximately 5 ml. of alcohol are added to the solution if part of it is to be kept for several days. Filtration is not necessary. The quantity of dithizone solution to be used for one day is shaken with about 100 ml. of dilute nitric acid (1 ml. per 100 ml. of solution) just before use. Standard Dithizone Solutions. These solutions are made from distilled chloroform which has been treated with hydroxylamine, as in the instructions for the dithizone extraction solution. In this case, however, the chloroform is filtered through a dry paper into a glass-stoppered Pyrex bottle, which is wrapped with heavy paper or kept in a wooden box t o protect it from light. The proper amount of dithizone (5 mg. per liter for the 0- to 10-microgram range, 10 mg. per liter for the 0- to 50-microgram range, and 20 mg. per liter for the 0- to 100-microgram range) is dissolved in the chloroform, absolute alcohol is added (5 ml. per liter), and the solution is ready for standardization. It should be kept in a refrigerator. Ammonia-Cyanide Mixture, Each liter of the mixture contains 20 grams of potassium cyanide and 150 ml. of distilled ammonium hydroxide (specific gravity 0.9), or its equivalent; it is brought to volume with double-distilled water. This solution should be kept in a cool place. Bufer Solution, pH 3.4. Reagent nitric acid (9.1 ml.) is diluted to approximately 500 ml. with double-distilled water in a 1-liter volumetric flask, bromothymol blue indicator is added, and the pH is brought to 3.4 with distilled ammonium hydroxide. Then 50 ml. of Clark and Lubs potassium acid phthalate-hydrochloric acid buffer, pH 3.4, double strength (50 ml. of 0.2 M potassium acid phthalate and 9.95 ml. of 0.2 M hydrochloric acid, per 100 ml.), are added and the solution is diluted to 1 liter with doubledistilled water. Chloroform Recovery. Used dithizone solution is collected in glass-stoppered amber bottles containing alcohol (about 0.5 per cent of capacity of bottle). Each liter of solution is washed with about 200 ml. of water, then washed with two 100-ml. portions of concentrated sulfuric acid. Five milliliters of alcohol are added to the chloroform and it is allowed to stand qvernight in the dark over lump calcium oxide. It is then distilled (through a still fitted with all-glass connections) into a glass-stoppered amber bottle containing absolute alcohol (about 1 per cent of capacity of bottle).
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Vol. 14, No. 11
Apparatus. Pyrex glassware should be used throughout; containers should all be glass-stoppered. The extractions are most conveniently carried out in Squibb-type separatory funnels, 150ml. capacity, graduated at 5-, lo-, and 10-ml. intervals to 100 ml., with the aid of rotary funnel racks (1). White vaseline is used to grease the stopcocks, since it is practically lead-free. Any photometer which can be used with the cells described is usually satisfactory for this procedure. A glass filter with maximum transmission at 510 millimicrons is suitable. The neutral wedge photometer described by Clifford and Wichmann ( 4 ) waa originally employed in this work. ISOLATION OF LEAD. Approximately 15 ml. of the ammonium citrate solution are poured into each of the lower funnels, using the graduation marks on the funnels as a guide. The proper aliquants of the prepared samples are next added (equivalent to 50 to 200 ml. of urine, 0.1 to 0.5 gram of fecal ash, 5 to 20 grams of blood, or one-tenth of a normal day’s food), together with 5 ml. of potassium cyanide solution and 1 ml. of hydroxylamine hydrochloride solution, and the mixtures are made alkaline to phenol red with distilled ammonium hydroxide. In rare instances the solutions will become very cloudy upon being made alkaline, because of the presence of an unusually large quantity of calcium phosphate; in such a case sufficient ammonium citrate solution should be added to dissolve the precipitate. The extraction of lead is started with 5 ml. of dithizone extraction solution and the color is noted in order that the proper standard dithizone solution may be chosen in the final lead estimation. (Usually the lead is not extracted quantitatively with each portion of dithizone solution because of the various salts present. Instead of 50 micrograms of lead, which is the theoretical equivalent of the dithizone, only 40 micrograms will be extracted, as a rule, by each 5 ml. When the quantity of lead present is less, than 10 micrograms this is recognized by the distinctive greenish-blue color of the dithizone.) Another 5-ml. portion of dithizone solution is added, using the graduation marks on the funnels as a guide. Before this combined 10-ml. solution is drained into the other funnel, its color is noted, for this should indicate whether the quantity of lead present is greater than 50 micrograms, or greater than 100 micrograms. The extraction is then continued with successive 5-ml. portions of dithizone solution, the color of each portion is noted, and each is drained before adding the next, until the lead is completely extracted. The combined dithizone extracts are washed with approximately 50 ml. of double-distilled water and the water is washed with 5 ml. of distilled chloroform. This chloroform wash should be green in color; if it is not, the presence of lead or zinc is indicated. A drop of potassium cyanide solution should then be added to the water and the funnel shaken again. if the chloroform phase does not become green the water shouid be washed a t least once with dithizone extraction solution. All chloroform washings are added to the dithizone extract and the water is discarded. The lead is stripped from the dithizone extract by shaking with 50 ml. of buffer solution (pH 3.4); if the dithizone solution does not return to its original green color, bismuth is present. The dithizone solution is drained from the funnel, and if the presence of bismuth has been indicated, the buffer solution is shaken with one 5-ml. portion of dithizone solution. After this is drained out, 5 ml. of distilled chloroform are added and the mixture is shaken. If the sample contains more than 100 micrograms of lead, as indicated by the color of the separate portions of dithizone extraction solution noted early in the lead isolation step, part of the buffer solution containing the lead is discarded before the 5 ml. of distilled chloroform are added. The quantity discarded should be sufficient t o reduce the lead content t o less than 100 micrograms; this is conveniently done by using the graduation marks on the funnel. The volume is then made up to 50 ml. by adding more buffer solution and the chloroform is added as indicated above. The funnel is allowed to stand unstoppered until the drop of chloroform floating on the surface of the aqueous phase has evaporated, and the 5 ml. of chloroform are drawn off as completely as possible without allowing water to enter the bore of the stopcock. FINAL ESTIMATIOX OF LEAD. In the following portion of the procedure direct sunlight should not be allowed to strike the solutions. The proper standard dithizone solution (10 ml. of the 0- to 10microgram solution, or 25 ml. of the 0- to 50- or 0- to 100-microgram solution) is added to the funnel containing the lead in 50 ml. of buffer solution. 7 ml. of ammonia-cyanide mixture are added, and the funnel ’is immediately shaken for 1 minute. The pressure which develops should not be released through the stopcock; instead, the stopper should be lifted in order to avoid blowing water into the funnel stem. If a number of analyses are bein run, the mixed color can be developed in the whole series an! photometric readings taken one after the other.
November 15, 1942
907
ANALYTICAL EDITION
Part of the dithizone solution (2 ml. of the 0- to 10-microgram solution, or 10 ml. of the 0- to 50- or 0- to 100-microgram solution) is used to flush the stem of the funnel; the end of the funnel stem is dried, and the solution is allowed to runfdirectly into the proper cell for the photometric reading. The 0- to 10microgram solution is used with a 5-cm. (2-inch) cell, the 0- to 50-microgram solution with a 2.5-cm. (1-inch) cell, and the 0- to 100-microgram solution with a 1.25-cm. (0.5-inch) cell. The cells are cylindrical, with optically plane ends and have an internal diameter of about 14 mm. Since the 0- to 10-microgram cell holds the entire 8 ml. remaining in the funnel, it cannot be rinsed with part of the dithizone solution, but must be cleaned and dried with pure acetone after each sample. The other two cells, however, can be rinsed with dithizone solution a t least twice, and it is rarely necessary to wash or dry them between sam les. Tge photometric readings are referred to calibration curves made for each standard solution in the following way: A standard lead nitrate solution is made by dissolving recrystallized lead nitrate in 1 per cent nitric acid, so that each milliliter contains 1 mg. of lead. From this solution two dilutions in 1 per cent nitric acid are made, one containing 10 micrograms of lead per milliliter, and the other 1 microgram per milliliter. All these solutions will keep indefinitely in glass-stoppered Pyrex containers. A standard lead solution in the proper buffer is made by taking a measured quantity of the desired standard lead solution, bringing it to pH 3.4 by addition of dilute distilled ammonium hydroxide, adding the proper amount of the Clark and Lubs buffer mentioned above, and diluting the mixture to a known volume. This solution should be made fresh each time it is used. Measured quantities of this standard lead solution are added to clean separatory funnels, the volume in each funnel is brought to 50 ml. with the pH 3.4 buffer solution, and the dithizone solution to be standardized is added. Color development and photo-
metric readings are then carried out as in the final estimation of lead. From these readings a standard calibration curve can be made.
Summary Certain modifications of the photometric dithizone method previously described are discussed. T h e observance of certain precautions makes i t possible t o keep standard dithizone solutions for months without apparent deterioration. The time required for carrying out an analysis has been decreased significantly b y the development of a lead-bismuth separation which permits the omission of the bismuth test. T h e complete analytical procedure, including preparation of samples and purification of all reagents-previously described in several papers-is given in detail.
Literature Cited (1) Bsmbach, Karl, IND.ENO.CHEM.,ANAL.ED., 11, 400 (1939). (2) Cholak, J., Ibid., 7, 287 (1935). (3) Cholak, J., and Story, R. V., Zbid.. 10, 619 (1938). (4) Clifford, P. A., and Wichmann. H. J.. J . Assoc. OfficialAer.
Chem., 19, 130 (1936). (5) Hubbard, D. M., IND.ENO.CHEY., ANAL.ED.,9, 493 (1937). (6) Kehoe, R. A., Thamann, F., Cholak, J., J . Znd. Hyg., 15, 257 (1933). ---, (7) Kluchesky, E. F., Longley, B. J., and Kozelka, F. L., J. P h r macol., 74, 395 (1942). (8) Smith, F. L., 2nd, Rathmell, T. K.,and Williams, T. L., Am. J. Clin. Path., 11,653 (1941). (9) Wichmann, H. J., IND.ENO.CHEW.,ANAL.ED., 11, 66 (1939). (10) Willoughby, C. E., Wilkins, E. S., Jr., and Kraemer, E. O., Ibid., 7,285 (1935).
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A Simple Apparatus for Small-Scale Catalytic Hydrogenation C. R. NOLLER
N
AND
hi. R. BARUSCH, Department of Chemistry, Stanford University, Calif.
UMEROUS designs of apparatus for small-scale cata-
lytic hydrogenation have been described in the literature, a good survey of which has been given recently b y Johns and Seiferle (f). W ile no claim is made for great originality in t h e apparatus now described, the authors believe t h a t they have combined t h e good features of several types of apparatus and modified them t o give one which is very simple in construction and operation (Figure 1). One of the inconveniences of most designs is the shaking mechanism, which usually is of the reciprocating type. Weygand and Werner (2) used an electromagnetic stirrer. T h e authors have substituted a medium-size Alnico permanent magnet for the electromagnet, and in place of the complicated devices for introducing the sample or catalyst have used a cup and stopcock. The type of buret used b y Johns and Seiferle (1) has been retained, since this avoids the use of a stopcock. As usually operated, the weighed sample to be hydrogenated is placed in the flask with a suitable solvent and the iron-cored stirrer. With the stopcock open, the flask is connected to the buret by the ground joint and held in placed with a buret clamp. The reservoir is lowered, the height of the magnet is adjusted, the stirrer is started, and the speed of the motor is regulated so
FIQURE 1. APPARATUS 50-cc. buret B. 50-cc. flask C. 19/38 interchangeable ground joint D. 5-cc. cup E . %om. section of 8 d . nail sealed in glass tubing F. Medium size Alnico magnet If. Brass saddle with setscrews L. 1/100 h. p. variable-speed motor M . Hydrogen inlet N . To reservoir for confining liquid A.
PL
