A TITRIMETRIC METHOD FOR THE QUANTITATIVE ESTIMATION OF LEAD IN BIOLOGICAL MATERIALS

BY M. K. HORWITT* AND GEORGE R. COWGILL

(From the Laboratory of Physiological Chemistry, Yale University School of Medicine, New Haven)

(Received for publication, March 31, 1937)

The announcement by Fischer in 1929 (1) of the remarkable affinity of dithizone (diphenylthiocarbazone) solutions for lead has stimulated many laboratories to search for practical micro- methods that could be applied to the determination of lead in biological materials. The theory and the application of such methods have been adequately described by Wilkins, Willoughby, Kraemer, and Smith (2), Ross and Lucas (3), and more recently by Clifford and Wichmann (4) and will not be treated further here.

The authors have had the opportunity to apply the various dithizone methods to a large variety of materials. Most of the published techniques have been critically investigated and this paper will present what is in our opinion the most satisfactory extraction procedure together with a new titration that eliminates the necessity of investing in expensive photometric equipment.

Interference by Bismuth and Tin-The red color produced by the reaction between solutions of dithizone in chloroform and those of a heavy metal in alkaline cyanide solution is not entirely specific for lead (5). Bismuth and stannous tin react with dithizone in a similar fashion and must be eliminated at some point in the analytical procedure. Bismuth may be present in biological speci- mens as a result of previous medication and stannous tin is not an uncommon constituent of the normal diet.

Fischer and Leopoldi (6), Winter et al. (7), and Tompsett and ilnderson (8) have made use of the fact that bismuth may be separated from lead by extracting the dithizone mixture with

* Lead Research Fellow.

553

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554 Lead in Biological Materials

solutions of alkaline cyanide. Clifford and Wichmann (4) have objected to this procedure on the grounds that the alkaline cya- nide extracted some of the lead along with the bismuth. Our investigations have shown us that the amount of lead extracted by washing with potassium cyanide solution varied not only with the concentration of the dithizone present but also with the con- centration of the potassium cyanide used. Thus the 1 per cent solutions of potassium cyanide caused an appreciable loss of lead (5 to 10 per cent), but if a 0.5 per cent solution is used, the loss of lead is negligible, if the extraction is properly conducted. Where comparatively large amounts of bismuth are present (25 times the quantity of lead or more), it is advisable to use the procedure proposed by Willoughby and associates (9) and extract the weakly acidified solution with an excess of dithizone. We suggest, however, that the aqueous solution containing the lead and bismuth be adjusted to pH between 3.0 and 3.5, instead of pH 2 as recommended by Willoughby et al., in order to obtain a more satisfactory separation. Since bismuth will react with dithizone to give a brown color to the chloroform, a large excess of bismuth is easy to notice.

It is not possible to make an efficient separation of stannous tin from lead by extracting an acidified mixture with dithizone, because the optimum pH for the reaction between stannous tin and dithizone is close to neutrality. Fortunately, stannic tin does not react with dithizone, and since the process of ashing con- verts the stannous tin to stannic tin, the former does not inter- fere. Small amounts of stannous tin can be separated from a chloroform solution of lead dithizonate by shaking with 0.5 per cent potassium cyanide. Any traces of tin which have not been oxidized or which have reverted to the stannous state are removed from the mixture by the cyanide solution.

Interference by Calcium Phosphate-The presence of large amounts of calcium phosphate, as in the analyses of bones, pre- sents a special problem, if one wishes to avoid using a sulfide precipitation. Winter and collaborators (7) have suggested a preliminary separation of the lead phosphate which they claim precipitates at a lower pH than calcium phosphate. Similarly, in the analyses of urine, Ross and Lucas (3) precipitate the calcium as the oxalate at pH 4.5, a procedure which entrains the lead and leaves most of the phosphate in solution.

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M. K. Horwitt and G. R. Cowgill 555

It has long been known that citrates exert a solvent action on the phosphates of calcium (10). In 1881, Terreil (11) showed that 7 gm. of calcium phosphate were dissolved by a solution of 100 gm. of citric acid which had been neutralized with ammonia. In analytical work it is not practical to use such large amounts of citric acid. Large concentrations of citric acid not only increase the specific gravity of the aqueous solution, thus slowing the chloroform separations, but also reduce the affinity of dithizone for lead so that large excesses of dithizone must be used to insure complete extraction. These disadvantages may be avoided, however, if proper adjustments are made between the volume of solution and the amounts of calcium phosphate and citrate used. Thus it was found that the lead in an aliquot equivalent to not more than 1.5 gm. of dry bone could readily be extracted from 350 cc. of solution at pH 8.0 containing 15 gm. of sodium citrate. A “wet digestion” with sulfuric, nitric, and perchloric acids was used during our preliminary work on lead methods, but was soon abandoned because of the large amounts of lead introduced by this acid mixture. Furthermore, the use of sulfuric acid com- plicated the analysis of materials high in calcium. Better results were obtained when “dry ashing” at a temperature below 500’ was used. In order to facilitate the preliminary charring of the biological materials, an overhead heater was devised by Nims and Horwitt (12), which greatly shortened the time required for an analysis. By application of radiant heat from above, a sample can be dehydrated and charred without danger of foaming or spattering. The resulting product may be placed directly into a muffle furnace at 475”.

A source of error in the analysis of lead may be found in the type of dish used for ashing when the quantities of ash are small. When 5 cc. portions of a lead nitrate solution containing 0.01 mg. of lead per cc. were evaporated in a porcelain dish and heated at 450’ for 2 hours, less than 80 per cent of the original lead was recovered by extraction with hot 20 per cent hydrochloric acid. Two extractions with alkaline citrate and two more with 10 per cent potassium cyanide accounted for another 15 per cent of the original lead. When the same experiment was repeated with silica dishes, the hydrochloric acid alone extracted 97 per cent or more of the added lead.

Interference by Iron-Dithizone solutions are oxidized by ferric

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556 Lead in Biological Materials

iron in the presence of cyanide and special precautions must be taken when blood or other materials containing much iron are analyzed. Wilkins and associates (2) resorted to a preliminary dithizone extraction in which the partial destruction of this re- agent was not important. Tompsett and Anderson (8) also used a preliminary extraction, except that in their case sodium di- ethyldithiocarbamate was used instead of dithizone. A simple procedure was suggested by Fischer and Leopoldi (13) who add hydroxylamine hydrochloride to prevent the oxidation of dithi- zone. This treatment is effective if the amounts of iron present are small.

Since ferric iron in the concentrations encountered does not oxidize dithizone in the absence of cyanide, Wichmann et al. (14) have suggested that less cyanide be used. Similarly, Cheftel and Pigeaud (15) reduced the amount of cyanide used and claimed that iron did not interfere with the extraction of lead under these conditions. In our own laboratory the best results were ob- tained when both hydroxylamine hydrochloride and reduced amounts of potassium cyanide were employed; this procedure permitted the analysis of larger amounts of blood and gave good results.

Principle of Titration-The method described below differs from other dithizone techniques in that it is not necessary to standardize the dithizone solution. The final titration is carried out .direetly with a dilute lead solution, thus eliminating the neces- sity for special precautions in the handling of the dithizone. The lead is separated from a given solution by means of dithizone and the resulting lead-dithizone complex is then isolated. The latter is freed of lead by washing with acid. The chloroform solution of dithizone remaining is mixed with some dilute cyanide solution which removes most of the dithizone from the chloroform, im- parting a brown color to the aqueous layer. A lead solution is added from a burette to this mixture until all the dithizone has been reconverted to lead dithizonate, as indicated by (1) the dis- appearance of the brown color in the aqueous layer, and (2) the absence of a red color when the aqueous layer is mixed with chloro- form and additional lead solution.

Apparatus-All glassware should, whenever possible, be made of Pyrex glass. Silica dishes are recommended for the ashing

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procedures. The cleansing of the separatory funnels before each analysis is especially important. Washing with hot dilute nitric acid followed by a thorough rinsing with water redistilled from an all-Pyrex still is usually sufficient for the separatory funnels, but the dishes in which materials have been ashed should be cleansed with the aid of a warm solution of alkali as well.

An overhead heater of some sort is recommended for preparing biological samples (12). Such a device not only shortens the time required to complete a determination but also affords protec- tion from laboratory dust during the diminished period of hand- ling. The muffle furnace used should preferably be equipped with a stainless steel sleeve to protect the samples from contamination by the brittle parts of the oven.

Reagents-The water should be distilled from an all-Pyrex still and all reagents should be stored in Pyrex containers.

1. Potassium cyanide solution. 10 gm. in 100 cc. prepared daily.

2. Hydrochloric acid, 20 per cent. Concentrated acid (sp. gr. 1.19) mixed with an equal volume of water and the mixture distilled from the all-Pyrex still.

3. Hydrochloric acid, 0.5 per cent. Prepared by diluting 25 cc. of Reagent 2 to 1 liter.

4. Chloroform, U.S.P. 5. Hydroxylamine hydrochloride solution, 25 per cent. 6. Dithizone solution. Dissolve 40 mg. of diphenylthiocar-

bazone in 400 cc. of chloroform and filter into a 500 cc. Pyrex separatory funnel. Add 50 cc. of water containing 2 cc. of 25 per cent hydroxylamine hydrochloride solution and shake. Keep in a cool dark place and withdraw the chloroform solution as needed. The acid aqueous layer not only prevents the oxidation of the dithizone but also extracts any lead which might be pres- ent. Further purification was not found necessary for the titri- metric method to be described below.

7. Potassium cyanide solution, 0.5 per cent. Prepared daily by diluting 25 cc. of Reagent 1 to 500 cc. It is important that this solution be lead-free. To insure this, place 100 cc. of Re- agent 1 in a separatory funnel and extract with 2 cc. of chloroform containing 2 drops of dithizone solution. If a pink color appears in the chloroform layer, withdraw it and repeat the extraction

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until the chloroform layer is colorless. The slight excess of dithi- zone which remains in the 10 per cent potassium cyanide is not significant, since the amounts which remain after dilution to form the.0.5 per cent solution are not detectable.

8. Nitric acid. Redistil the concentrated reagent. 9. Ammonium hydroxide. Distil the concentrated reagent

into cold redistilled water. 10. Standard lead solutions. Dissolve 1.599 gm. of recrystal-

lized lead nitrate (or the equivalent of lead acetate) with the aid of 1 cc. of nitric acid and dilute to 100 cc. This solution which con- tains 10 mg. of lead per cc. is quite stable. By diluting 10 cc. of this solution to 100 cc. and then in turn diluting 10 cc. of the latter to 1 liter, a solution containing 0.01 mg. of lead per cc. is prepared. This is stable in Pyrex glass containers for at least 5 days.

11. Sodium citrate, 20 per cent. To 800 cc. of this solution add 8 cc. of 10 per cent potassium cyanide and extract in a 1 liter separatory funnel with 15 cc. portions of dithizone solution until the citrate mixture is free of lead. Wash twice with 25 cc. por- tions of chloroform, acidify with 4 cc. of 20 per cent hydrochloric acid, and complete the extraction of the excess dithizone with 20 cc. portions of chloroform.

Procedure

Blood-A 10 cc. sample in a 50 cc. silica evaporating dish is dried and completely charred beneath a radiant heater. This is accomplished in approximately 45 minutes. Transfer to a muffle adjusted to a temperature of about 475”. After 2 hours, remove from the muffle, moisten with 2 cc. of nitric acid, and place the dish beneath the radiant heater until the reaction has ceased and the material is free of excess acid. This requires approximately 30 minut,es. Return to the muffle for about half an hour to com- plete the oxidation,

Place the dish on a hot-plate, carefully add 15 cc. of 20 per cent hydrochloric acid, and heat until the ash is dissolved. Wash the contents into a 125 cc. separatory funnel with about 20 cc. of hot water. Add 10 cc. of 20 per cent sodium citrate and 3 cc. of ammonium hydroxide to the silica dish, mix, and transfer to the separatory funnel with enough water to make a total volume

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of about 75 cc. Cool, add 1 cc. of hydroxylamine hydrochloride, 1 drop of phenol red, and bring to pH 8.0 with ammonium hydrox- ide delivered from a Pyrex burette. Cool, add drop by drop, shaking between additions, 0.5 cc. of 10 per cent potassium cyanide, and immediately extract with 0.5 cc. of dithizone solu- tion and 4 cc. of chloroform. If, after shaking, the chloroform layer does not contain a noticeable excess of uncombined dithizone, add 0.2 cc. portions of dithizone solution, shaking between addi- tions, until the green excess becomes evident. Remove the chlo- roform phase to another separatory funnel and repeat the extrac- tion of the aqueous phase twice with 0.2 cc. portions of dithizone in 2 cc. of chloroform. To the combined chloroform solutions add an amount of 0.5 per cent potassium cyanide equal to 1.5 times the volume of the chloroform solution and shake for 10 seconds. Withdraw the chloroform layer to another separatory funnel and wash the aqueous cyanide solution with 1 cc. of chloro- form. Combine the chloroform solutions and again extract with 1.5 volumes of 0.5 per cent potassium cyanide solution. Any lead which may have dissolved in the aqueous phase is re- moved by extraction with 2 cc. of chloroform.

The extraction with cyanide solution described above removes the uncombined dithizone unless a very large excess has been used, in which case the extraction with 0.5 per cent potassium cyanide is continued until the absence of color in the aqueous layer indi- cates that the dithizone excess has been removed. The lead is separated from the red dithizone complex by shaking for 15 seconds with 2 volumes of 0.5 per cent hydrochloric acid. With- draw the green chloroform layer and then extract the acid aqueous solution with 1 cc. of chloroform to recover the last traces of dithi- zone. Combine the chloroform fractions.

Titration-Add to the dithizone solution 0.5 volume of 0.5 per cent potassium cyanide and shake. Most of the dithizone goes into the aqueous layer, giving that mixture a brown color. Add the standard lead solution (0.01 mg. per cc.) from a burette a drop at a time, shaking between additions, until only a very faint color remains in the aqueous phase. ‘This is evidence that practically all of the dithizone has combined with lead and gone into the chloroform layer. Discard the red chloroform phase and wash the aqueous layer with chloroform, 2 cc. at a time, until the chloro-

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form layer remains colorless after shaking. (In order to prevent any loss of uncombined dithizone the color in the cyanide solu- tion should not be greater than that color which 0.2 cc. of Re- agent 6 will impart to 10 cc. of 0.5 per cent potassium cyanide solution.) Add a drop or two of the lead solution and shake for 5 seconds. Withdraw the pink chloroform solution and continue the extraction with 2 cc. portions of chloroform plus a drop or two of lead solution until further addition of lead gives no pink color to the chloroform solution after shaking. The end-point is a slight pink in the chloroform solution; extraction with 1 more drop results in a colorless solution. In order to facilitate the titration, a solution of the lead-dithizone complex containing a small amount (1 or 2 drops) of the lead solution in 2 cc. of chloroform is kept for comparison. When the color obtained after an addition of lead solution is less than that given by 1 drop of lead, the end- point has been attained. It is suggested that the beginner add 2 drops (about 0.0006 mg.) at a time until his eyes become accus- tomed to the change.

Urine-Measure 200 cc. of urine into a 250 cc. silica dish and ash as described above for blood, except that 5 cc. of nitric acid should be used instead of 2 cc.

Because the ash of urine is sometimes difficult to dissolve, moisten it with 15 cc. of 20 per cent hydrochloric acid and heat until almost dry. Transfer the contents to a 500 cc. separatory funnel with the aid of an additional 15 cc. of hydrochloric acid, 50 cc. of 20 per cent sodium citrate, and 5 cc. of ammonium hydrox- ide. ,Make to a volume of 250 cc. and cool. Add 1 drop of phenol red and slowly bring to pH 8.0 with ammonium hydroxide. Add 3 cc. of 10 per cent potassium cyanide; extract with an excess of dithizone using 0.5 cc. portions in 3 cc. of chloroform and pro- ceed as with blood beginning with “TO the combined chloroform solutions add. . .”

Bone-Place a known amount of dried bone in a silica dish and heat in a muffle at 475” for 2 hours. Remove, add an amount of nitric acid equivalent to 3 cc. for each 1.5 gm. of dried bone, and evaporate to dryness beneath the radiant heater. Return to the muffle for about 3 hour.

Dissolve the contents of the silica dish, using 15 cc. of 20 per cent hydrochloric acid for each 1.5 gm. of dried bone, and transfer

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an aliquot containing not more than 1.5 gm. to a 500 cc. Pyrex separatory funnel. Add 75 cc. of sodium citrate solution, 2 drops of phenol red, and enough water to make a volume of about 350 cc. Add ammonium hydroxide 1 drop at a time, shaking and cooling during the addition, until pH 8.0 has been attained. Add slowly 5 cc. of 10 per cent potassium cyanide and extract the clear solution with an excess of dithizone. Add 3 cc. of dithizone solu- tion and 3 cc. of chloroform, and shake for 30 seconds. If the chloroform layer is not purple, add 1 cc. of dithizone solution at a time until the purple color remains after shaking, indicating that a large excess of dithizone has been used. Good results are ob- tained in the presence of large quantities of citrate, if a 100 per cent excess of dithizone is used at this stage. Withdraw the chloroform layer to a 125 cc. separatory funnel and extract the aqueous mixture three times with 1 cc. of dithizone solution plus 2 cc. of chloroform, 0.5 cc. of dithizone plus 2 cc. of chloroform, and 3 cc. of chloroform, respectively. Combine the chloroform solutions and extract once with 2 volumes and twice with equi- volumes of 0.5 per cent potassium cyanide. Remove the lead from the lead-dithizone complex with 2 volumes of 0.5 per cent hydrochloric acid and titrate the resulting dithizone solution as described under blood beginning, “Add to the dithizone solution 0.5 volume. . .”

Blank Determination-A separate blank is run for each type of material analyzed. It is also important that an amount of lead approximately equivalent to that which one is likely to ob- tain from the material be added to the reagents when the blank is estimated in order that the blank determination may serve as a daily check on the technique of the analyst. This permits the use of as much dithizone as in the regular procedure and may bring out errors of manipulation that might not otherwise be caught. Thus, if 0.02 mg. of lead is added to the reagents and the final titration shows that 0.0223 mg. was present, the blank for reagents for a determination between 0.01 mg. and 0.03 mg. of lead would be 0.0023 mg.

Notes on Procedure

1. A good grade of white Vaseline is used for stop-cocks. 2. Care must be taken during the separation of the liquid

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562 Lead in Biological Materials

phases that no drops of the chloroform layer remain on the surface of the aqueous layer or adhere to the sides of the funnel.

3. If a precipitate should appear in the alkalinized solution of the ash just prior to the extraction of the lead with dithizone, redissolve the suspension with hydrochloric acid and add more sodium citrate before again alkalinizing.

4. It is sometimes more satisfactory to extract the aqueous layer with more chloroform rather than wait for the last traces of chloroform to separate out.

5. A small chloroform trap should always be maintained in the separatory funnel, rather than to attempt a complete separation.

EXPERIMENTAL

Accuracy of Fundamental Titration-Inasmuch as the final measurement in this method involves a determination of an un- known quantity of dithizone in terms of its combination with lead, it is necessary to prove the accuracy of such a titration. This was done as follows:

Measured quantities of a dilute solution of dithizone were trans- ferred to a separatory funnel, diluted with chloroform to 15 cc., mixed with 10 cc. of 0.5 per cent potassium cyanide, and titrated with lead nitrate solution. The average time for each titration was approximately 5 minutes. The results as given in Table I indicate that the sensitivity of the titration is about 0.0002 mg.

Accuracy of Method Applied to Biological Materials-Repre- sentative results for the recovery of different amounts of lead added to blood, urine, and bones are given in Table II. The amounts added correspond to the lead which may be found in normal and pathological states. The accuracy of the method for blood is about 10 per cent, for urine and bones about 3 per cent.

A large number of foods and diets have been analyzed for lead during the past year, and we find it preferable not to use a definite procedure in these cases because of the varying amounts of iron and calcium in different products. Instead, the method is modi- fied to suit the material. Thus, animal rations which may be high in calcium are treated by the technique described under bone. Should the material contain a considerable amount of iron, care is taken to use some hydroxylamine hydrochloride and a minimum of the 10 per cent potassium cyanide solution before extraction.

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The analyst is safe, whatever procedure is used, provided a blank determination accomplished in a similar manner gives a good recovery.

TABLE I

Titration of Dithizone with Lead Nitrate

Dithirone PbNO, (0.01 III&!. Per cc.) Lead nitrate used for each cc. of dithizone

cc. cc. cc.

1 0.52 0.52 3 1.56 0.52 3 1.54 0.51 6 3.14 0.52 6 3.13 0.52

10 5.18 0.52 10 5.18 0.52

TABLE II

Recovery of Lead from Blood, Urine, and Bones

Material

10 cc. beef blood

200 cc. human urine

1.5 gm. bone ash*

-

Lead in

ml.

0.0016

0.0060

0 .O,l28

-

“8.

0 .OOlO 0.0020 0.0030 0.0040 0.0050 0.0100 0.0200 0.0300 0 .0400 0.0500 0.0100 0.0300 0.0500 0.1000 0.1500

-

mg. w. 0.0025 0.0009 0.0036 0.0020 0.0047 0.0031 0.0060 0.0044 0.0066 0.0050 0.0163 0.0103 0.0265 0.0205 0.0358 0.0298 0.0455 0.0395 0.0560 0.0500 0 .0230 0.0102 0.0429 0.0301 0.0637 0.0509 0.1128 0.1000 0.1618 0.1490

* Aliquots of an acid solution of bone ash.

DISCUSSION

T

ElT0r

mg.

0 .OOOl 0.0000 0 .OOOl 0.0004 0.0000 0.0003 0.0005 0.0002 0.0005 0.0000 0.0002 0.0001 0.0009 0.0000 0.0010

I

-

-

tWOVery

per cent

1::

103 110 100 103 103 99 99

100 102 100 102 100 99

The dithizone methods published to date depend upon the standardization, at some time or other, of the dithizone solution used. Since the ultimate standard is a lead solution, a method

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which does not depend on the concentration of the dithizone and makes direct use of a lead standard instead has many advantages. Furthermore, since the dithizone complexes fade on standing, a method in which use of a 10 cc. burette is substituted for a colorimet- ric procedure should prove more accurate. Another advantage of the method is that the amounts of bismuth and tin which are present in biological specimens are eliminated by the regular pro- cedure.

Each determination of the blank serves not only as a daily check on the skill of the operator, but also on the efficacy of the method. The short period of heating described is important in the prevention of loss of lead; therefore, care should be exercised not to prolong the ashing procedure.

For routine analyses of foods in a control laboratory, it is prob- ably sufficient to titrate to the disappearance of the brown color in the potassium cyanide solution.

SUMMARY

A quantitative method for the determination of lead based on a new titrimetric procedure is described. Results of the applica- tion of this method to biological materials are reported.

BIBLIOGRAPHY

1. Fischer, H., 2. angew. Chem., 42, 1025 (1929). 2. Wilkins, E. S., Jr., Willoughby, C. E., Kraemer, E. O., and Smith,

F. L., Ind. and Eng. Chem., Anal. Ed., 7, 33 (1935). 3. Ross, J. R., and Lucas, C. C., J. Biol. Chem., 111, 285 (1935). 4. Clifford, P. A., and Wichmann, H. J., J. Assn. Off. Agric. Chem., 19,

130 (1936). 5. Fischer, H., Mikrochemie, 8, 319 (1930). 6. Fischer, H., and Leopoldi, G., Z. angew. Chem., 47, 90 (1934). 7. Winter, 0. B., Robinson, H. M., Lamb, F. W., and Miller, E. J., Ind.

and Eng. Chem., Anal. Ed., 7, 265 (1935). 8. Tompsett, S. L., and Anderson, A. B., Biochem. J., 29, 1851 (1935). 9. Willoughby, C. E., Wilkins, E. S., Jr., and Kraemer, E. O., Ind. and

Eng. Chem., Anal. Ed., 7, 285 (1935). 10. Nicholls, J. R., Analyst, 66, 594 (1931). 11. Terreil, M. A., Bull. Sot. chim., 36, 548 (1881). 12. Nims, F. L., and Horwitt, M. K., Znd. and Eng. Chem., Anal. Ed., 8,

275 (1936). 13. Fischer, H., and Leopoldi, G., Z. ges. exp. Med., 97,819 (1936). 14. Wichmann, H. J., Murray, (3. W., Harris, M., Clifford, P. A., Loughrey,

J. H., and Vorhes, F. A., Jr., J. Assn. 08. Agric. Chem., 17,108 (1934). 15. Cheftel, H., and Pigeaud, M. L., Ann. fuls., 29, 76 (1936).

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M. K. Horwitt and George R. CowgillLEAD IN BIOLOGICAL MATERIALS

QUANTITATIVE ESTIMATION OF A TITRIMETRIC METHOD FOR THE

1937, 119:553-564.J. Biol. Chem. 

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