JULY
llj,
1935
ANALYTICAL EDITION
normal1,y expected. I n Table V are given the average limits of accuracy which were obtained from an average of the results on the different portions of each range. On range 1 (in the central portion) the six operators varied from 0 to 0.1 p. p. m. of sulfate. I n the lower portion of range 2 , they varied from 0.1 to 0.2 p. p. m. of sulfate and in the upper, from 0.4 to 0.7 p. p. m. of sulfate. For higher sulfate content, covered by ranges 3 and 4, the following variations were incurred: in the lower portion of range 3, from 0 to 2.0 p. p. m. and in the upper from 1 to 5 p. p. m.; in the lower region of range 4 from 0.5 to 4 p. p. m. and in the upper 1.0 to 6 p. p. m.
Conclusions The results obtained with this method compared favorably with those obtained by gravimetric analysis. Kone of the ions or mixtures of ions, with the exception of iron, exerted any appreciable influence on the method. This influence of iron was due to color and was corrected by running a blank on the unprecipitated sample. A varnation in temperature between 17” and 32” C. had no effect on the determination. Different batches of the precipitating agent, BaC12.2Hz0,also were found to exert no determining influence on the method. Sulfate concentrations above the direct range of the instru-
265
ment can be obtained by diluting the sample and multiplying by the appropriate factor. The method is rapid, a determination taking less than 10 minutes, and strictly applicable to boiler and feed-water studies where accurate low sulfate determinations are desirable. The investigators feel that there is a possible application in other fields for sulfate determination.
Acknowledgment The authors wish to acknowledge the financial assistance and sponsorship of W. H. & L. D. Betz, who made this investigation possible. The kindness of the Hellige Company of New York is acknowledged in furnishing the instrument for this investigation.
Literature Cited (1) Parr, S., and Staley, D., 1x11.ENG.CHEM.,Anal. Ed., 3, 66-7 (1931). (2) Schroeder, W. C., Ibid., 5, 403-6 (1933). (3) Treadwell, F. P., and Hall, W. T., “Analytical Chemistry,” 1701. 11, p. 401. RECEIVED April 27, 1935. Preaented before the Division of Water, Sewage, and Sanitation Chemistry a t the 89th Meeting of the American Chemical Society, New York, N. Y., April 22 to 26, 1935. J
Determination of Lead A Modification of the Fischer-Leopoldi Method 0. B. WINTER, HELEN M. ROBINSON, FRANCES W. LAMB, AND E. J. MILLER, Michigan Agricultural Experiment Station, East Lansing, Mich.
HE recent r e g u l a t o r y measures of the U n i t e d States F o o d a n d Drug Administration limit the a m o u n t of lead that may be present in s p r a y r e s i d u e s on f r u i t , m d necessitate the development of methods which are capable of determining minute quantities of lead and which a t the same time are less cumbersome and time-consuming than those hitherto available. Within the past 2 years a number of methods have been proposed to fill this need, including a colorimetric sulfide method by Wichmann arid Vorhes ( 6 ) , an electrolytic method by Wichmann and Clifford (6),a colorimetric dithizone method by Vorhes and Clifford (4)based on the method of Fischer and Leopoldi ( d ) , and a photometric method by Samuel and Shockey (3). I n an a t t e m p t to use the d i t h i z o n e (diphenylthiocarbazone) colorimetric m e t h o d for lead in the routine examination of s p r a y r e s i d u e s , difficulties were encountered and a different modificaicion of t h e F i s c h e r -
T
A colorimetric method based on the use of dithizone (diphenylthiocarbazone) for the microdetermination of lead is described. The lead is extracted from solution by means of a chloroform solution of dithizone, the intensity of the resultant red color of the lead dithizone compound in chloroform solution is measured, and the corresponding quantity of lead is read from a curve constructed from measurements made on known amounts of lead. The method is applicable to the estimation of lead in spray residues and biological materials and is sensitive to approximately 0.001 mg. in these materials. Slight modifications of the method permit determination of lead in the presence of the interfering elements, bismuth and stannous tin. The method has the advantage of being both extremely sensitiv? and relatively rapid. With spray residues as many as 36 determinations may be made in one day, and with biological materials approximately 20, depending upon the type of material and method of preparing samples for analysis.
Leopoldi method was developed and has been used for the past two seasons in the analysis of the residue on nearly a thousand samples of apples. It is the purpose of this paper to describe the method. The F i s c h e r - L e o p o l d i (2) method is based on the following properties of dithizone (1): 1. Dithizone is insoluble in water or dilute a c i d b u t v e r y soluble in chloroform or carbon tetrachloride, giving a green color to the solution. 2. A dilute ammoniacal solution dissolves dithizone a n d extracts it from the chloroform or carbon t e t r a c h l o r i d e , giving a brownish color to the a q u e o u s solution. 3. Ammonium c i t r a t e decreases the solubility of dithizone in a dilute ammoniacal solution. 4. Lead salts in aqhueous solution when shaken wit dithizone dissolved in chloroform or carbon t e t r a c h l o r i d e r e a c t with the dithizone to form a highly colored cherry-red compound s o l u b l e in chloroform. This lead dithizone compound is not extracted from chloroform or carbon tetrachloride by dilute ammonium hydroxide. A dilute acid solution, however, decomposes the
266
INDUSTRIAL AXD ENGINEERING CHEMISTRY
lead dithizone compound and extracts the lead but leaves the free dithizone quantitatively in the chloroform or carbon tetrachloride. Fischer and Leopoldi extracted the lead in the presence of potassium cyanide with two or three portions of carbon tetrachloride-dithizone solution and removed the excess of dithizone by means of two or three extractions with dilute ammonia. Then with d i l u t e a c i d they d e c o m p o s e d the lead dithizonecompound, t h e r e b y removing the lead from the carbon tetrachloride a n d l e a v i n g the free dithizone quantitatively in the carbon tetrachloride. T h e green dithizonecarbon tetrachloride solution was made to v o l u m e with carbon tetrachloride and compared colorimetrically with a standard prepared in an identical manner a s t h e sample. They s t a t e t h a t the dithizone solution must be fresh; that the presence of lead causes the color t o change FIGURE! 1. CURVERELATINGCOLORfrom green to red IMETER READINGS TO MILLIGRAMS OF LEAD almost instantly; t h a t a curve which shows the relation between the quantity of lead extracted and the intensity of the green color produced after the lead dithizone compound hasbeen decomposed is linear; that in the presence of potassium cyanide the only heavy metals that interfere with the reaction are stannous tin, thallium, and bismuth; that a quantity of lead as small as 0.0001 mg. in a drop of solution can be detected; and that their experimental error was 3 to 5 per cent when determining lead present in the amount of 0.01 per cent and 5 t o 8 per cent in amounts less than 0.001 per cent. I n the authors' study of the underlying principles of the Fischer-Leopoldi method it was found that a much shorter procedure could be used without the sacrifice of accuracy, and even with greater accuracy and fewer possible sources of error. Contrary to finding a linear relationship between the intensity of the green color of the dithizone and the lead content, the authors found that the relationship between the intensity of the red color of the lead dithizone solution and the lead content was not linear, but could nevertheless be advantageously used for the colorimetric determination of the amount of lead present. In the first place this eliminated the step of decomposing the lead dithizone and removing the lead, which saved considerable time and possibility of error in manipulation, and furthermore the red color is more easily compared in the colorimeter and is more stable than the green color of the dithizone itself. I n some cases the red dithizone lead standards have been found to be unchanged after more than a month and the chloroform dithizone solution reagent requires no other precaution for its preservation than the
VOL. 7, NO. 4
customary brown-glass container used for the chloroform itself. Sufficient reagent was usually prepared for 3 or 4 weeks' use. It has also been found unnecessary in this procedure to purify the dithizone powder before making up the reagent. The dithizone used was procured from Eastman and from two other independent sources. Further saving of time was effected when it was found that with proper precautions one extraction of the sample for the removal of lead gave as good results as when two or three extractions were made, and only two extractions were ordinarily necessary for the removal of the excess dithizone.
Reagents Solution A. Chloroform, c. P. Solution B. Ammonium citrate, 5 per cent solution prepared from citric acid by making- slightly - - alkaline with ammonium hydroxide. Solution C. Potassium cvanide. 5 Der cent solution. Solution D. Dithizone, l"0 mg. dissolved in 400 cc. of chloroform. Solution E. (Lead-free reagent 1). Add 30 cc. of solution C, 15 cc. of B, and 5 cc. of concentrated ammonium hydroxide to approximately 450 cc. of distilled water. Solution F. (Lead-free reagent 2). Add 10 cc. of solution C and 5 cc. of concentrated ammonium hydroxide to about 500 cc. of distilled water. Solution G. (Standard lead solution). Dissolve 1.5985 grams of recrystallized lead nitrate in 100 cc. of 0.1 per cent nitric acid (1 cc. = 0.01 gram of lead). Dilute 0.5 cc. of this solution to 500 cc. (1 cc. = 0.01 mg. of lead). Solutions E and F should be tested for the absence of lead as follows: Place 20 cc. in a small separatory funnel, add 1 cc. of dithiaone and 5 cc. of chloroform, shake, separate, and extract the chloroform layer once or twice with another 20 cc. of the solution. The final chloroform layer should be colorless or very faintly red. (The faint red may persist if separatory funnels made from ordinary glass are used. A correction for this minute amount of lead, however, is included in the blank which must always be run on the reagents used in the preparation of the sample for analysis.)
If solutions E and F are not free from lead, they should be treated as follows: To solution E add a few cubic centimeters of dithizone (solution D) and about 15 cc. of chloroform and shake vigorously for 5 minutes. Allow a t least 5 minutes for the layers to separate. If the chloroform layer is red and the water layer colorless, add more dithizone and repeat the shaking and the separation. Draw off the chloroform layer. Repeat the above procedure, adding about 1 cc. of dithizone and 10 cc. of chloroform each time until the chloroform layer is practically colorless or slightly green. This layer should become colorless or very faintly red when extracted once or twice with solution F. For solution F proceed as in the preparation of solution E. (In this solution, no more dithizone should be added than is necessary to remove all the lead. The water layer should have only a slightly brownish color immediately after preparation and should become entirely colorless after standing several hours. The solution should not be used until it is colorless.) The brown color may also be rcmoved by shaking out with 0.25 gram of ash-free decolorizing charcoal, thus avoiding the necessity of waiting for the solution to decolorize. If charcoal is used, it is not necessary to avoid the addition of an excess of dithizone.
Procedure EXTRACTION OF LEAD FROM SAMPLE.Place approximately 15 cc. of solution in a 150-cc. Squibb separatory funnel and add a neutralized aliquot of the sample. From a buret add a sufficient amount of dithizone solution in 1-cc. portions so that, after
a few vigorous shakings and allowing the layers to separate for a few seconds, the lower layer has a noticeable purple color, indicating the presence of an excess of dithizone. Add chloroform (also from a buret) to make a total volume of dithizone and chloroform exactly 10 cc. Shake and allow the layers to separate, Give the funnel an occasional slight swirling motion and a gentle horizontal shaking while the layers are separating, in order to cause the drops of chloroform which collect at the top of the aqueous layer to fall to the bottom. Draw off the chloroform layer into a second separatory funnel in which 20 CC. of
JULY 15, 1935
ANALYTICAL EDITION
solution F have been placed. Discard the solution remaining in the first funnel. EXTILACTION OF EXCESS DITHIZONEFROM CHLOROFORM LAYER. Shake the second separatory funnel and allow the layers to separate as before. Separate and repeat the extraction and separation (occasionally a third extraction is necessary) with 20 cc. of solution F until the upper solution is practically colorless, indicathg that the dithizone has all been removed. PREP'ARATION OF LEAD DITHIZONE SOLUTIONFOR READINQ IN COLORIMETER. Open the stopcock of the separator funnel slightly until the chloroform replaces the water in t l e bore; then by means of a cotton swab remove the moisture from the stem of the funnel. Draw off the clear or slightly turbid chloroform layer into a dry test tube, retaining in the separatory funnel any small amount of emulsion which may have formed and collected :it the chloroform-water interface. (Since the dithiaone solutiort and the chloroform are measured accurately from burets and the amount of chloroform retained in the water layer when running the sample undoubtedly is approximately equal to that in the standard, and since any chloroform which forms an emulsion with the water presumably retains a proportional amount' of lead, no chloroform should be added to correct any loss in the volume of the finished sample. The small amount of emdsion which sometimes persists should, therefore, be retained ::n the funnel.) Stopper t'he test tube and, if necessary, allow it to stand for several hours for cloudiness to disappear. Cloudiness is sometimes due to the formation of a fine emulsion of aqueous solut'ion in the chloroform during the shaking, which may persist for some little time. It also appears when the chloroform solution cools below the temperature at which the separation occurs. In the latter case it may be removed by warming the solution very slightly by holding the test tube in the palm of the hand. Compare in a colorimeter with a standard. The standards are prepared in a similar manner as the unknown. Since the intensity of the color is not directly proportional to the lead. content, it is necessary t o make up a series of standards and ploi a curve from which to read the quantity of lead present. CONSTRUCTION OF GRAPH. In order to construct a standard curve, determinations were made on four series of standard lead solutions. Each series consisted of ten samples containing 0,0010,
267
colorimeter readings fall on the most sensitive part of the curve, the use of Pyrex separatory funnels, double-distilled water of very low lead content, etc., it should be possible to increase the accuracy still further. Since the method is extremely sensitive, it is essential that precautions be taken to eliminate all sources of contamination of lead. All glassware should be cleaned with nitric acid before use, and reagents with the smallest possible quantitites of lead in them should be selected. Stopcocks should be lubricated with lead-free lubricants; a pure white vaseline was found satisfactory for this purpose.
TABLE11. RESULTSOF DETERMINATIONS o s SOLUTIONS OF KNOWNLEADCONTENT Colorimeter Readings
Lead Present
Lead Found
54.5 44.0 32.8 32.5 32.6 29.0 23.8 23.7 23.7 17.8 16.8
Mg. 0.0070 0.0080 0.0120 0.0125 0.0125 0,0135 0.0165 0.0176 0,0175 0,0225 0.0250
Mg. 0.0067 0,0084 0,0120 0,0120 0.0121 0.0134 0.0167 0.0169 0.0169 0.0228 0.0250
Error MI?. -0.0003 +O ,0004
0.0000 -0.0005 -0.0004 -0.0001 +0.0002 -0.0006 +O -0.0006 ,0003
0.0000
% -4.3 S5.Q 0.0 -4.0 -3,2 -0.7 +1.2 -3.8 -3.5 +1.3 0.0
PROCEDURE FOR ROUTINE WORK. When making a large number of determinations it is advantageous to run a t least six samples simultaneously. An adjustable rack holding twelve separatory funnels (Figure 2) was found very convenient for this purpose. The following procedure works to good advantage :
Place 15 cc. of solution E in each of the six separatory funnels in,the lower row. Add a neutralized aliquot of the sample and mix. (If a large number of samples are to be analyzed-as, for example, spray residue solutions in which the acidity of the samples is fairly uniform--solution E should be made up with sufficient ammonium hydroxide so that the quantity of ammonium hydroxide in 15 cc. is slightly in excess of that necessary to practical they were calculated to this value. neutralize the acid in a 5-cc. sample. This eliminates the necessity of neutralizing each individual sample or of adding ammonium hydroxide to solution E in each separatory funnel.) The i~esultsare shown in Table I, which gives the amount Add the dithizone and chloroform to each of t$ funnels, as of lead present in the solution, the corresponding colorimeter directed under "Extraction of lead from sample, and shake. readings, and the averages of these readings. Figure 1 Place 20 cc. of solution F in each funnel in the upper row. Draw off the chloroform layer from the first funnel into the funnel shows the averages of the readings in Table I expressed in the directly above and discard the remaining water layer. Shake form of a graph. Once this graph has been constructed it the upper funnel. Proceed in the same way with each of the may be used indefinitely, but the 0.0200 standard is made up other five funnels. Place 20 cc. of solution F in each of the lower daily in routine work. row of funnels and proceed as before. (If the proper amount of dithizone has been added, two extractions will be sufficient to TABLEI. RELATIONBETWEEN QUANTITYOF LEADPRESENT remove the excess of dithizone and the water layer will be practically colorless; if not, another extraction will be necessary.) AND COLORIMETER READINGS Remove the water from the stems of the separatory funnels by Colorimeter Readings means of a cotton swab. Draw off the chloroform layers into Lead Series Series Series Series test tubes and stopper the tubes. The solutions are now ready Present 1 2 3 4 Average to be read in the colorimeter. Mo .
0.0025, 0.0050, 0.0100, 0.0150, 0.0200, 0.0250, 0.0300, 0.0350, and 0.0400 mg. of lead, respectively, and each was read against 0.0200 mg. of lead as the standard. (When an extremely small amount of lead is to be determined, a curve should be plotted with 0.0500 mg. of lead as the standard.) The colorimeter readings were made with the standard set at 20, or when this was not
0 . 0010 0.0'025 0.0050 0.0100 0.0150 0.0200 0.0250 0.0300 0.0350 0.0400
160 120 67.8 38.2 25.2 20.0 16.4 14.5 13.1 11.8
165 117 69.0 38.0 26.4 20.0 16.8 14.8 13.3 12.2
155 119 69.2 38.6 26.8 20.0 16.9 15.0 13.0 12.1
iii'
68.4 38.2 26.0 20.0 16.5 14.6 12.9 12.2
160 119 68.8 38.2 26.1 20.0 16.7 14.7 13.1 12.1
ACCURACY OF THE METHOD.Determinations were made on a large number of solutions containing known amounts of lead added as lead nitrate in order to determine the accuracy of the method. In Table I1 are presented the results of a number of representative analyses, showing the colorimeter readingcr, the amount of lead present, the amount found by analysis, and the percentage error. With only ordinary care the maximum error can be kept below 5 per cent. By the exercise of greater care and attention to details, such as choosing the size of the sample to be analyzed so that the
Factors Influencing Rapidity and Accuracy of Method Satisfactory methods in control work must necessarily require as little time as possible for their manipulation, sometimes a t the sacrifice of extreme accuracy. Therefore in order to make the procedure as short as possible and maintain the desired accuracy, it appeared necessary to study and determine the following: (1) the number of extractions necessary t o remove all the lead from the sample; ( 2 ) the quantity of dithizone necessary to extract all thelead from the sample (this is important because if a large excess is added extra washings are necessary for its removal); (3) the time required for shaking in each extractive operation; (4) the number of extractions necessary to remove the excess of dithizone from the chloroform layer; ( 5 ) the possibility of error due to solubility of the lead dithizone compound in the
INDUSTRIAL AND ENGINEERING CHEMISTRY
268
VOL. 7, NO.
The possibility of error due to the solubility of the lead dithizone compound in solution F under the conditions of the extraction was determined by reextracting the lead dithizonechloroform layer, after it was ready for the colorimeter reading, witb two additional 20-cc. portions of ammoniacal solution F, and taking the colorimeter reading after each extraction. These results are shown in Table IV. BY ADDITIONAL EXTRACTIONS WITH TABLEIV. LEADREMOVED SOLUTION F
.4fter second extraction
Lead Present
ammoniacal solution; and (6) the quantity of solvent lost by volatilization or solubility and the effect of this loss on the accuracy of the method. To determine the number of extractions necessary to remove all the lead from the sample, each of a number of solutions of known lead content was extracted three times with an excess of dithizone and chloroform. The quantity of lead found in each extraction is shown in Table 111. TABLE111. LEADREMOVED BY SUCCESSIVE EXTRACTIONS WITH DITHIZONE 7
Lead Present
MQ. 0.0050 0.0100 0.0200 0.0250 0,0300 0.0350 0.0400
First extraction
MQ* 0.0052 0.0100 0,0195 0.0238 0.0285 0.0350 0.0400
Lead Found Second extraction MQ.
0,0002 0,0002 0.0003 0.0005 0.0007 0.0003 0.0001
--.
Third extraction
MQ. 0.0000 0.0001 0.0000 0.0001 0.0002 0.0001 0.0000
Although the data in Table I11 do not take into consideration the solubility of lead dithizone in solution F, they show that only a small amount of lead mas recovered after the first extraction of the sample and that only one extraction of the sample with the dithizone-chloroform solution is necessary. In order to determine the quantity of dithizone necessary to extract all the lead from solution, a number of synthetic lead solutions were extracted with different quantities of dithizone-chloroform reagent. The color of the chloroform layer was taken as the criterion for determining the quantity of dithizone needed. Such quantities of dithizone were taken that, after the extraction, the color of the chloroform layers varied from pure red through different combinations of red and green to a large excess of green. The results indicated clearly that when sufficient dithizone was added so that, after shaking, the chloroform layer showed positive evidence of green mixed with the red giving rise to a reddish purple, practically no lead remained in the aqueous solution. Experience has shown that a thorough shaking of each sample for 15 seconds is sufficient time for each extraction of the lead from the sample and for each extraction for the removal of the excess dithizone from the chloroform layer. The excess dithizone in the chloroform layer which does not combine with the lead must be removed completely. This is accomplished by extracting the chloroform layer two or three times with the ammoniacal solution F until the ammoniacal layer remains colorless. If the excess of dithizone in extracting the lead from the sample is kept low, two extractions of approximately 20 cc. each are sufficient.
After second additional extraction
MO.
M Q.
0.005 0.005
0.0047 0.0044
0.0044 0,0042
0.0040 0.0039
0.0100 0.0100
0,0099
0,0095
0.0091 0,0092
0.0069 0.0082
0 0150 0.0150
0,0147 0.0158
0.0139 0,0143
0.0134 0.0136
0,0200 0,0200
0,0204 0.0200
0,0204 0,0204
0.0194 0.0200
0.0250 0.0250
0,0255 0.0260
0,0250 0.0260
0.0240 0.0240
0.0300 0.0300
0.0290 0.0305
0.0290 0.0290
0.0275 0.0285
MQ.
FUNNELS FIGURE 2. ADJUSTABLERACKFOR SEPARATORY
Lead FounAfter, first additional extraction
MQ.
The data in Table IV show that lead dithizone is appreciably soluble in solution F and no more extractions should be made than necessary to remove the excess dithizone. This emphasizes the fact that the volume of the solution from which the lead is to be extracted and also the volume and number of the wash solutions must be kept as uniform as possible, If the above precautions are taken, however, with both the samples and the standard, no serious error results from the solubility factor. The quantity of solvent lost during the procedure of a determination, except where a n appreciable amount of emulsion is formed and discarded, is approximately 0.4 cc. This loss, however, is the same in both the standard and the samples, and again there appears to be no considerable source of error here. OF RESULTS OF LEADDETERMINATIONS TABLEV. COMPARISON BY “LONG”AND “SHORT”PROCEDURES
Lead Analyst Sample Present
Lead Found by Long Method
&fQ.
MQ.
Error
Mo*
Lead Found by Short Method
Error
-0.0001 -0.0006 -0.0003 0.0000 0.0000
A
1 2 3 4 5
0.0050 0,0100 0.0150 0.0200 0.0300
0.0050 0.0098 0.0148 0.0200 0.0300
0.0000 -0.0002 -0.0002 0.0000 0.0000
MQ. 0.0049 0,0094 0.0147 0.0200 0.0300
B
6 7
0.0070 0.0120 0.0200 0,0220 0.0270 0.0320
0.0073 0.0118 0.0200 0.0218 0.0263 0.0324
-0.0003 -0.0002 0.0000 -0,0002 -0.0007 -0,0004
0.0072 0.0118 0.0200 0.0228 0.0266 0.0320
8
9 10 11
MQ.
t 0.0002
-0.0002 0.0000
to.ooos
to.0004
0.0000
For the purpose of comparing the accuracy of the procedure described earlier in this paper with that in which more extractions are made, several series of known lead solutions were analyzed by making two extractions of the samples with dithizone and three extractions for the removal of the excess dithizone, and a standard curve was plotted. A number of known solutions were then run by both procedures. Table V gives a comparison of the results by the above procedure (Lilong”)with those by the procedure described earlier in this paper (“short”), made by two different analysts. These data indicate that the results by the “short” method are comparable with those by the r‘long” method. From the preceding studies it appears that one extraction
JULY 115, 1935
ANALYTICAL EDITION
with a slight excess of dithizone is sufficient for the removal of the lead from the sample, and that two extractions are sufficient for the removal of the excess dithizone from the chloroform layer, provided the proper amount of dithizone is used for extraction of the lead. TABLEVI. EFFECTOF INCREAS~NG THE CONCENTRATION OF AMMONIUM HYDROXIDE IN SOLUTIONS E AND F Lead Present
Lead Found la
Me. 0 0200
0 0200
Mg.
0.0199 0.0200
2b Mg. 0.0196 0.0184 0.0200 0.0200
0.0200 0.0200 0.0206 0,0204 0.0210 0.0200 0 Solutians E and F containing 5 cc. of NHaOH per 500 cc. of solution. b Solutions E and F containing 15 cc. of NHcOH per 500 cc. of solution.
I n order to show the effect of increasing the ammonium hydroxtde concentration of the solution from which the lead is extracted and of the solution used for extracting the excess dithizone (solutions E and F), lead determinations were made on known solutions using solutions E and F, and also using solutions E and F to each of which had been added an extra 10 cc. of concentrated ammonium hydroxide per 500 CG. of solution. The results of these determinations are shown in Table VI, and indicate that the increase in the quantity of ammonium hydroxide, within the limit given, does not affect the recovery of the lead.
Estimation of Lead in Spray Residues on Apples The samples of apples used in this work were stripped of their residue by the method of Wichmann and Vorhes (6). Each apple was impaled on a pointed glass rod and swirled in
a boiling sodium oleate-sodium hydroxide solution for approximately 1 minute or until the skin began to break. It was then washed with hot 1 per cent acetic acid squirted on from a short-
stemmeld 25-cc. pipet fitted with a rubber bulb to insure satisfactory uniform washing and keeping the volume within desired limits, and the dipping and wash solutions were combined and made up to a volume of 500 cc. with distilled water. A 100-cc. aliquot of this was added to 10 cc. of concentrated nitric acid to precipitate the soap and wax, filtered, and a suitable aliquot (1 to 5 cc.) of the filtrate was used for the determination of the lead. An aliquot should be taken so that the quantity of lead present will fall on the more sensitive portion of the curve. Other spray residue solutions Cere prepared according to the method of Wichmann and Clifford (5). The fruit was peeled, an aliquot of the peelings was digested with nitric acid, and the aoid solution was made to a volume of 500 cc. An aliquot of this wmi taken for the determination. When a 1400-gram sample of apples is taken, the spray residue solution made to 500 cc., and 5 cc. of this solution are taken for the determination, the milligrams of lead read from the curve in Figure 1 are numerically equal to twice the number of grains of lead per pound of fruit. I n the authors' work the sodium oleate-hydroxide dip method, as previously described, was found most satisfactory and was adopted as routine practice. Approximately 1400 grams were taken for each sample and a 3-cc. aliquot was finally taken for the analysis. Using these quantities the lead could be determined fairly accurately as low as approximately 0.002 and as high as 0.035 grain per pound of fruit. I n case the red color persisted after adding about 3 cc. of dithizone and shaking, in the procedure where the lead was extracted from the sample with the dithizone solution, the sample was discarded and a smaller aliquot was taken. The quantity of lead as read from the curve was corrected for the blank and calculated to grains per pound of fruit by the following formula:
2@
(Reading from curve minus blank) 2
I -
11
5
= cc. of aliquot taken X 10
1400
gram of fruit grain of lead per pound of fruit
For example, if a 1300-gram sample was taken, the volume of the solution was made to 500, 100 cc. of this were added t o 10 cc. of concentrated nitric acid, a 3-cc. aliquot was taken, the lead found as read from the curve was 0.0200 mg. and the blank for 3 cc. was 0.0030 mg., then 1400 5 11 (o'0200 - 0'0030) X __ X - X - = 0.0168 (grain per pound) 1300
2
3
10
Lead determinations were made on a large number of spray residues by the colorimetric method herein described and checked by the electrolytic method of Wichmann and Clifford (6). The close agreement of the results by the two methods is indicated by representative data in Table VII. TABLEVII. DETERMINATIONS OF LEADIN SPRAY RESIDUEB BY ELECTROLYTIC AND COLORIMETRIC METHODS
-
Spray Residue Solution Electrolytic Gr./lb. 1038 0.0044
Lead Found Colorimetric Gr./lb. 0,0058 0,0058
Difference Gr./lb. $0.0014 + O . 0014
1039
0.0045
0,0042 0,0042
- 0.0003
1027
0.0105
0.0103 0,0103
-0.0002 -0 IO002
1033
0.0126
0,0119
-0.0007
0.0145
0,0134 0,0134 0,0130
-0.0011 -0.0011 -0.0015
501 1032
0.0148 0.0179
0 0136
-0 0012 -0.0005
850
0.0383
0,0370 0,0380
-0.0003
0,0583 0.0575
$0.0029 $0.0021
1025
1031
0.0554
0.0174 0.0174
-0,0003
-0.0013
After some practice with the colorimetric method, an analyst can make from 30 to 36 lead determinations in 8 hours on spray residue solutions after the solutions have been prepared for analysis by either of the methods used and made to volumes of 500 cc. This includes the time necessary for the preparation of the solutions for extraction, the extractions, the colorimeter readings, and the calculations, but not the weighing and dipping of the fruit, which can be done by helpers or assistants. During the past season the procedure has been used by a control laboratory, apparently with no diffi. culty and with results concordant with the authors'.
Application to Animal and Plant Materials Some work has been done on the determination of lead in biological materials. The data here reported are the results obtained on one or two samples of each kind of material and are intended simply to show the application of the method to these materials. If the sample is a solid, place from 1 to 5 grams, depending on the amount of lead present in a porcelain crucible and add 1 cc. of sulfuric acid (1 to 10). Add sufficient distilled water to moisten the sample completely, and mix thoroughly. Dry in an oven at 100' C. Burn in a muffle at about 500" C. If the sample is a liquid add the sulfuric acid, evaporate t o dryness, and proceed as with a solid. Moisten the ash in the crucible, add 1 cc. of concentrated hydrochloric acid, and place the crucible on a steam bath for several minutes. Add 2 cc. of nitric acid (1 to 5), transfer to a small beaker, and make just alkaline t o litmus paper with ammonium hydroxide. Add 2 cc. of 5 per cent citric acid and again make
INDUSTRIAL AND ENGINEERING CHEMISTRY
270
TABLEVIII. LEADIN ANIMALAND PLANT MATERIALS Material
Quantity Lead Taken Found
cc.
0.0030 0.0040 0.0040 0.0036 0.0038
Blank on reagents, plus 0.0200 mg. of lead
0.0225 0.0246 0.0252 0.0248 0.0240
Beef blood 1
10
Beef blood12
10
25
Grams
Corn
1
0,0099
Wheat
1
0.0067 0.0056 0.0062
0 . 5 0.0030 0.0032 Alfalfa
0.0110 0.0112 0.0108 Apple peels (oven-dried) 0.0108 0.0026 0.0027 0.0028
0.0046 0.0050 0.0047 0.0044 0.0052
0.0096 0.0092
Human urine 1
Quantity Lead Taken Found Cc. Mg.
Material
MQ.
Carrots (oven-dried)
1
0.0025
0.5
0.0074 0.0081
2
0.0168 0.0164
MQ. Blank
0
The following statements should be made regarding the use of the method for the determination of lead in such materials as bone meal, fish meal, meat scraps, etc., which contain a very high percentage of calcium phosphate. This large amount of phosphate cannot be held in solution when the sample is made alkaline unless so small an aliquot is taken that the results are of no value. Some preliminary work was done on this kind of material and it was found that lead phosphate precipitates a t a lower p H than calcium phosphate. If ammonium hydroxide is added slowly, while stirring vigorously, to the acid solution of the above-mentioned and other similar materials until a slight permanent precipitate forms, the lead, iron and aluminum phosphates and a small amount of calcium phosphate will be precipitated. Filter this but do not wash. Discard the filtrate and dissolve the precipitate on the filter with dilute hydrochloric acid and collect the solution in the same beaker in which the phosphates were precipitated. Wash very thoroughly with hot water. (Special care must be taken to dissolve all of the phosphate that adheres to the sides of the beaker.) Proceed as directed under plant and animal materials.
0.0024
TABLEIX. LEADFOUND IX BOXEMEAL Lead Added
VOL. 7, NO. 4
Lead Found Mg 0.0030 0.0027 0.0032
.
Lead was determined in 2 grams of bone meal by this method. After burning and dissolving the meal, the solution was made to a volume of 200 cc. and 10-cc. aliquots were taken (0.1 gram) for analyses. The results are shown in Table I X and indicate that added lead can be recovered quantitatively by the above method and that duplicate and triplicate results are in close agreement; hence the method should be applicable to materials high in calcium phosphate.
Interfering Elements
Bismuth,’stannous tin, and thallium combine with dithizone dissolved in chloroform and like lead form intensely colored solutions. Bismuth forms a brown color, stannous 0.0100 0.0276 Bone meal plus blank tin a red color of a different tint than that of lead, and thal0.0280 lium a cherry-red color which cannot be distinguished from 0.0200 0,0400 Bone meal plus blank lead. If any of these elements are present with the lead in 0.0400 the ammoniacal citrate-cyanide solution from which the lead is to be extracted they will be extracted with the lead. ForTABLE X. DETERMINATION OF LEADIN PRESENCE OF B I S M ~ T R tunately, however, the bismuth and stannous tin dithiaone AND STANNOUS TIN compounds are soluble in solutions E and F containing an exLead Lead Element cess of ammonium hydroxide. Hence, if 10 cc. of concenPresent Found Present MB. Mg. MQ. trated ammonium hydroxide are added to each 500 cc. of Bismuth solutions E and F and the samples are treated in the usual 0.0200 0.0196 0.0100 manner, the bismuth and stannous tin are removed while 0.0200 0.0207 0.0100 0.0200 0.0204 O.OlO0 the lead remains. When there is a large amount of bismuth 0.0200 0.0197 0.0100 0.0200 0.0196 0,0500 present it is usually necessary to make an extra extraction 0.0200 0.0197 0.0500 with solution F. Table X shows that, with the above changes Tin in the procedure, lead may be determined accurately in the 0.0200 0.0201 0.0050 0.0200 0.0218 0.0100 presence of these elements. 0.0200 0.0100 0.0200 Unfortunately thallium is not soluble in solution F and hence when present interferes with the determination of lead. An attempt was made to use carbon tetrachloride instead alkaline with ammonium hydroxide. If a precipitate forms of chloroform as a solvent for the dithizone for extracting the which does not redissolve on heating, add hydrochloric acid droplead. Lead combines with dithizone when dissolved in carwise until the solution is clear (except for a possible small amount of insoluble silicates), and 2 cc. in excess. Again make just bon tetrachloride as well as when dissolved in chloroform, and alkaline with ammonium hydroxide. (Any precipitate that the excess of dithizone is removed more easily by the ammoforms on making the solution alkaline occludes the lead and hence niacal solution from the carbon tetrachloride layer than from must be redissolved and held in solution. This may be accomplished by adding a large excess of sodium or ammonium chloride the chloroform layer. However, the lead dithiaone compound t o the solution before neutralizing. If this does not prevent the is slowly extracted from the carbon tetrachloride layer by the precipitate from forming, a smaller sample of the original maammoniacal solution and furthermore is not so stable as in terial should be taken for the analysis.) Add the solution to chloroform solution, and therefore carbon tetrachloride canthe separatory funnel containing solution E, and determine the lead as previously directed. not be used for accurate quantitative determinations. Bone meal plus blank
0
0.0180 0.0188 0.0190
Table VI11 gives the results of lead determinations on a few of these materials, and shows that when a lead salt was moistened with sulfuric acid and burned in a muffle a t about 500’ C., the lead was recovered quantitatively, and that fairly consistent results were obtained in determining lead in a few animal and plant materials.
Aclcnowledgment The authors wish to express their appreciation to W. C. Dutton and L. R. Farish of the Horticultural Department for furnishing the samples of apples and preparing them for analysis.
ANALYTICAL EDITION
JULY 15,1935
Literature Cited Fischer, H., Wiss. Verdfentlieh. Siemens-Konwn, 4, 158 (1925); 2. angew. Chem., 42, 1025 (1929). ;2) Fischer, H., and Leopoldi, G., Wiss. Verofentlich. SiemensKonzern, 12, 44 (1933). :3) Samuel, B. L., and Shockey, H. H., J. Assoc. 0ficial Agr. Chem., 17, 141 (1934).
(1)
271
(4) Vorhes, F. A,, Jr., and Clifford, P. A., Ibid., 17, 130 (1934). (5) Wichmann, H. J., and Clifford, P. A., Ibid., 17, 123 (1934). (6) Wichmann, H. J., and T'orhes, F. A., Jr., Ibid., 17, 119 (1934). RECEIVED May 31, 1935. Presented before the Division of Physical and Inorganic Chemistry a t the 88th Meeting of the American Chemical Society, Cleveland, Ohio, September 10 t o 14, 1934. Published with the permimion of the director of the Experiment Station as Journal Article 212 (n. 8.).
Oxidation-Reduction Indicators I. Diphenylbenzidine Sulfonic Acid L. A. SARVER AND WM. VON FISCHER School of Chemistry, University of Minnesota, Minneapolis, Minn.
I
T HAS already been pointed out ( 2 ) that diphenylbenzi-
dine disulfonic acid is an intermediate product in the oxidation of diphenylamine sulfonic acid to its violet colored holoquinoid form, and that it should possess all the advantages of the latter indicator, yet require a much smaller correction when used with dilute oxidizing solutions. The authors, accordingly, have tried a number of possible means of synthesis; but only the direct sulfonation of diphenylbenzidine gave any promise of success. This reaction proceeds vigorously, and one or more of the poly-sulfonic acids may be easily obtained.
Prt:paration of Sodium Diphenylbenzidine Sulfonate One gram of dry powdered diphenylbenzidine, prepared as described in a recent paper ( I ) , is added little by little t o 10 ml. of 60 per cent fuming sulfuric acid in a shallow dish over a period of about 2 minutes. The mixture heats up, but should not be allowed to become warmer than 40" C . As complete solution as possible is obtained by stirring and mashing up any lumps with a rod; then, after not more than 3 minutes, it is poured onto ice. Difficulty in controlling the reaction makes it undesirable to work with larger quantities, but the products of as many portions as desired may be combined at this point, and diluted t o a volume of 200 ml. for each gram of diphenylbenzidine used. The holution is then heated t o boiling, and the green color reduced t o brown with a stream of sulfur dioxide; the excess of the latter is removed by boiling, and any charred material filtered off. The filtrate is carefully neutralized with sodium carbonate, diluting if an precipitation occurs, and the sodium sulfate rendered insolubye by the addition of four volumes of ethyl alcohol; the sodium diphenylbenzidine sulfonate is appreciably soluble in 80 per cent alcohol, while all but an extremely small amount of the sodium sulfate is precipitated. After filtering, the residue should be redissolved in water, and reprecipitated with alcohol as often as necessary t o secure complete removal of the yellow color; the total volume of the combined filtrates is usually about 5 liters for each gram of diphenylbenzidine used. The alcohol is boiled off rapidly, and the evaporation completed on a steam bath, avoiding long exposure or overheating of the residue, which should be a light yellow. The yield is 3 to 4 grams per gram of diphenylbenzidine used.
Composition of Sulfonated Product Microscopic examination showed no appreciable quantity of sodium sulfate to be present; moreover, the known insolubility of this salt in 80 per cent alcohol precludes such a possibility. Chemical analysis indicated that ten sulfonate groups had been added in most cases; products prepared with 25 per cent fuming sulfuric acid contained four sulfonate groups. Exact information as to the positions of the substituted groups in the molecule would be difficult to obtain, and
is not necessary for the present purpose. Presumably, several different poly-sulfonic acids might be produced under different experimental conditions. STOCKSOLUTION OF INDICATOR. A 0.1 per cent solution of the sodium diphenylbenzidine sulfonate in water is recommended; this can be kept for several months, but not indefinitely. One drop or even less of this solution is satisfactory for titrations with 0.01 or 0.001 N solutions; 10 drops are preferable when 0.1 N solutions are used.
Indicator Properties As might be expected, diphenylbenzidine sulfonic acid strongly resembles diphenylamine sulfonic acid in most of its properties. When small amounts are oxidized in 0.5 to 1.0 N sulfuric acid solution, the pale yellow becomes first deep yellow, then green, and finally violet. Upon reduction, the color changes are reversed. It works perfectly in the presence of tungstate, and its color development is extremely sensitive to the catalytic effect of traces of ferrous iron, when dichromate is used as the oxidant. I n such a case, backtitration with ferrous solution, after treatment with an excess of oxidant, is more reliable; the error involved by even 10 minutes' contact with a reasonable excess of dichromate is negligible. For accurate microanalysis, the indicator corrections should be determined experimentally, but they are approximately equal to the volumes of indicator used, in terms of a 0.001 N solution. ABSORPTION SPECTRA. When diphenylbenzidine sulfonic acid is fully oxidized by dichromate, permanganate, or ceric sulfate, reddish violets are produced which are stable, but which fade after a long time. Bromine yields blues which fade very rapidly. The half-oxidized green form, on the other hand, is extremely stable, and does not precipitate out on standing, as was the case with that resulting from diphenylamine or diphenylbenzidine. Absorption curves were determined with a Keuffel and Esser color analyzer, the violet form giving one similar to that for diphenylamine sulfonic acid violet (a), with a maximum a t 5600 A,, while the green has a minimum a t this point, and absorbs strongly a t both ends of the visible spectrum.
Literature Cited (1) Sarver, L. A., and Johnson, J. H., J. Am. Chem. SOC.,57, 329 (1935). (2) Sarver, L. A., and Kolthoff, I. M., Ibid., 53, 2902 (1931).
RECEIVED March 25, 1935.
