INDUSTRIAL
and
ENGINEERING
CHEMISTRY +
ANALYTICAL EDITION
Harrison E. Howt?, Editor
Determination of Iodine In Thyroid and Its Preparations by Cerate Oxidimetry WAYNE W. HILTY
AND
DALE T. WILSON, Eli Lilly and Company, Indianapolis, Ind.
first hydrates and with continued stirring in the cold completely dissolves and is ready to be standardized as follows: Transfer 2 cc. of standardized ferrous sulfate solution to a 250-cc. beaker, using a calibrated pipet, with 2 M sulfuric acid or hydrochloric 0.025 M o-phenanthroline ferrous compl unknown ceric sulfate solution. T h e en pale green. An overstepped end point ca change, slightly green t o pink). $ *P volume of 0.1 N FeSOc X 20 X Of Ce(S04)2 = volume of Ce(S04)2required normality of FeS04
I
N AN attempt to develop a procedure which would be superior to that in the U. S. Pharmacopeia XI for the determination of iodine in thyroid preparations, the methods ,
presented in the literature b y Middleton (4),Kendall (d), and Harrington and Randall (I), involving fusion with an alkali in the presence of an oxidizing agent, and b y Trevorrow and Fashena (7), incorporating oxidation in an acid medium, were investigated. In the former methods the solutions of the fusion mass are neutralized and made acid, after which an excess of either chlorine or bromine is added for the purpose of oxidizing any iodide t o the iodate form. In the authors' opinion, inconsistent results, obtained by these methods, are due to technical difficulties in eliminating these excess oxidizing agents. The latter method proved to be unsatisfactory for routine analysis. Further search of the literature revealed the more recent works of Lewis (3) o n the determination of iodides by ceric sulfate and of Smith (6, 6) on the volumetric oxidation by ceric sulfate and the application of o-phenanthroline as an indicator in this reaction.
This solution has been found to remain stable for periods of at least 2 months. FERROUS SULFATE,0.1 N . Dissolve 27.80 grams of ferrous sulfate (FeS04.7HzO) in 980 cc. of water to which have been added 20 cc. of dilute sulfuric acid (one volume of concentrated sulfuric acid plus one volume of water). Standardize as follows: Take 25- or 50-cc. portions of the approximately 0.1 N solution of ferrous sulfate with a calibrated pipet and transfer to a 250-cc. beaker. Dilute to 200 cc. with 2 M sulfuric or hydrochloric acid. Add 1 drop of 0.025 M o-phenanthroline ferrous complex and titrate with standard potassium dichromate until the pink solution turns pale green. An overstepped end point can be backtitrated (color change, pale green to pink).
Experimental Normality of FeS04 =
The authors' recent experimental work reveals t h a t when thyroid gland or a mixture of thyroid gland and a diluent is fused with anhydrous sodium carbonate both inorganic and organic iodine combines with sodium to form sodium iodide. Since this iodide salt and the unused sodium carbonate are readily soluble in water, the insoluble carbonaceous material can be removed by filtration. T h e resulting solution, when acidified with hydrochloric acid t o make a 2 M (molar) solution of the acid, can be titrated with volumetric ceric sulfate and the percentage of iodine contained in the original thyroid sample calculated. The sodium iodide formed in the reaction described above is quantitatively oxidized by the ceric sulfate solution according t o the following equation: NaI
+ 6Ce(SO4)2 + 3H20.. . . HC1 . ... 9
.
+
NaIOs 3CedSOds
volume of 0.1 N K2Cr20, required volume of FeS04 required
x 0.1
POTASSIUM DICHROMATE, 0.1 N . Dissolve 4.9035 grams of reagent potassium dichromate, which has been pulverized and dried to constant weight a t 120' C., in sufficient distilled water to measure exactly 1000 cc. a t standard tem erature. 0-PHENANTHROLINE FERROUS SULFATE~OLTJTIOS, 0.025 M . o-Phenanthroline monohydrate, molecular weight 198, melting point 90-100' C. Use 1.485 rams for 100 cc. of 0.025 M solution as below. Ferrous suffate, 0.025 M solution Use 0.695 gram of ferrous sulfate heptahydrate in 100 cc. of soiution. Make fresh as needed. Dissolve the ferrous sulfate, add the o-phenanthroline monohydrate, and stir until all is dissolved, giving a dark red solution. One drop of this indicator serves for each titration in ~1 volume of approximately 150 cc. HYDROCHLORIC ACID, reagent grade.
Procedure for Thyroid Gland
+ 3HzsO1
Thoroughly mix 1 gram of thyroid, finely powdered and accurately weighed, with 15 grams of anhydrous sodium carbonate in a nickel crucible of about 125-cc. capacity, and spread an additional 10 rams of anhydrous sodium carbonate evenly over the surface. b e a t the crucible in the flame of a Bunsen burner at a rate to attain a dull red color in 10 minutes. Then place the crucible and contents in a muffle furnace and heat at a temperature not to exceed 500" C . for 30 minutes. Cool the mixture and transfer it to a 250-cc. beaker containing 100 cc. of warm distilled water. Rinse the crucible with 25 cc. of dis-
Reagents ANHYDROUS SODIUM CARBONATE, reagent grade.
CERIC SULFATESOLUTION, 0.005 N . Dissolve 1.70 to 1.80 grams of anhydrous ceric sulfate [Ce(S04),, molecular weight 332.253 in 1000 cc. of cold, dilute sulfuric acid made by adding 300 cc. of dilute sulfuric acid (one volume of water plus one volume of concentrated sulfuric acid) to 700 cc. of water. The salt 637
INDUSTRIAL AND ENGINEERING CHEMISTRY
638
tilled water and add it to the beaker. Apply gentle heat to the beaker and contents to ensure solution of the sodium carbonate and iodide. Filter the solution while still warm and wash the carbonaceous material with several small portions of warm distilled water. Cool the filtrate and cautiously neutralize with concentrated hydrochloric acid, using litmus paper as an indicator. For each 100 cc. of neutralized solution, add 20 cc. of concentrated hydrochloric acid and titrate with 0.005 N ceric sulfate, using a micro-
VOL. 11, NO. 12
buret and 1 drop of o-phenanthrdine ferrous sulfate, 0.025 M solution, as indicator, the end point being the first bluish green tinge that remains in the solution for 1 minute. Conduct a blank test with the same quantities of the same reagents omitting only the thyroid, and fusing as directed, and subtract the volume of 0.005 N ceric sulfate consumed from that consumed by the thyroid. Each cubic centimeter of 0.005 N ceric sulfate is equivalent to 0.0003178 gram of iodine in thyroid combination.
Procedure for Thyroid Tablets OF RESULTS FOR THYROID GLAKD TABLE I. COMPARISON
(U7eight of samule, 1.0000 gram) 0.005 N 0.005 N Ce(S04)z NarS!O3 Sample Requireda Requireda
Assay Method
A A
Proposed
u. 9. P. X I
.. .. ..
A
A
... ... ... ...
0,1949 0.1955 0.2035 0.1971 0,2019
20.22 21.50
0.214 0,228
...
5 733
u. s. P. X I 4
B B
%
...
6.132 6.151 6.403 6.203 6.363
A A A
Iodine
cc .
cc.
u. I W Z
5.653
...
... ...
21.26 20.04
0.1797 0 225 0,212
Weigh not less than 20 of the tablets and reduce them to a fine powder without an appreciable loss. Substitute approximately 1 gram of tablet mixture, accurately wei hed, for t h e thyroid sample and proceed with the proposed a n t U. S. P. XI methods for the assay of thyroid gland. The sample weight should be increased so that the amount of thyroid will equal or exceed 259.2 mg. (4 grains). Percentages of the labeled amount found are calculated on the basis of 0.200 per cent iodine in the thyroid gland.
Accuracy of Method A mixture consisting of four parts of potassium iodide (99.40 per cent KI) and one part of lactose, having a theoretical iodine content of 60.78 per cent, was assayed b y both the U. S. P. XI and the proposed methods for thyroid. Results obtained are listed in Table v.
Blank previously deduoted.
Comments and Conclusions
Fusion of the thyroid material with anhydrous sodium OF RESULTS FOR 6.48-MG. (O.~-GRAIX) TABLE 11. COMPARISON THYROID TABLETS carbonate does not destroy the carbonaceous material. HowPer Cent of ever, this does not interfere with the solubility of the sodium Thyroid 0.005 N 0.005 N Iodine Labeled Assay in Ce(S,O,)z NazSzOa per Amount of iodide formed in the reaction. Attempts to incorporate an Method Sample Requireda Required" Tablet Thyroid oxidizing agent with the anhydrous sodium carbonate to deGrains CC. Cc. MQ. stroy the carbonaceous material proved unsatisfactory, in Proposed 4.00 1.649 ... 0.001310 100.46 0.001324 101.56 4.00 1.667 ... that i t interfered with the ceric sulfate titration. 4.00 1.722 .... , . 0,001368 104.90 4.00 1.740 0.001382 106.00 The sodium chloride formed when the excess sodium car4.00 1.676 ,< . .: 0,001332 102.10 bonate is neutralized with hydrochloric acid does not interfere 4.00 2.50 2.50
1.640
.
I.,
.'
0.001303 0.000973 0.001100
99.91 75.10 84.90
with the oxidation of the sodium iodide. Sulfuric and nitric acids, if used to neutralize the excess sodium carbonate, give 4 Blank previously deduoted. salts which are easily oxidized b y ceric sulfate and thus give high results. OF RESULTS ON MANUFACTURERS' TABLE 111. COMPARISON The data as listed in the tables show the comparable results SAMPLES OF 64.8-MG. GRAIN) THYROID TABLETS that may be obtained by the two methods. The proposed Per Cent of method has the advantage of titrating the original iodide Thyroid 0.005 N 0.005 N Iodine Labeled Assay in Ce(S0a)l NazSzOa per Amount of formed in the fusion reaction and thus does not introduce any Method Sample Sample Requireda Requireda Tablet Thyroid other steps which are likely to interfere with the final result. Grains Cc. cc. MQ. The proposed method is applicable for the gland as well as Proposed A 10 4.149 ... 0,01319 101.74
u. s. P.
XI
U.5. P. X I Propd
...
2.300 2.600
10 10 10 10 10 12 12 12
4.240 4.348 4.155 4.043 3.983
B B
10 10 10 10 10 10
4.092 4.072 4,131 4,102 4,013 4.033
B
B B
12 12 10 10 10 10 10 10 15 U.S.P. X I c 15 C 10 10 10 10 10 12.72 U. 8. P. XI D 12.72 D a Blank previously dedwted. L
.
A A A A A A A A
B
U.8. P. XI
...
,
,.
,..
...
., , .,. ., , ., , ..,
14.80 16.00 14.58 , , , , , ,
,..
,..
,.. ,..
16.25 16.10
B B
4.112 4.171 4.161 4,289 4.250 4.230
... ...
4.112 4.072 3.993 4.191 4.103
... ...
... ... ... ...
... 19.97 19.49
... 17.59 18.22
0,01348 0,01382.0.01321' 0.01285,. 0.01166 0,01305 0,01411 0.01285 0.01300 0,01294 0,01313 0,01303 0,01275 0.01282 0.01433 0.01420 0.01307 0.01326 0.01322 0.01363 0,01351 0,01344 0,01409 0.01375 0.01307 0.01294 0.01269 0.01332 0.01304 0.01463 0.01515
102.95 106.62 101.79 99.14 97.67 100.70 108.80 99.19 100.34 99.85 101.30 100.59 98.40 98.89 110.50 109.50 100.83 102.2s 102.13 105.17 104.22 103.73 108.60 106.10 100.83 99.85 97.91 102.77 100.61 112.10 116.90
TABLE IV. COMPARISON OF RESULTSFOR 129.6-MG. @-GRAIN) THYROID TABLETS Thyroid 0.005 N 0.005 N Assay in Ce(S.04)~ NarSzOa Method Sample Requireda Requireda Grains CC. cc. Proposed 10 3.894 , ,. 10 3.954 , 10 3.875 . 10 3.865 10 3.944 ... 10 3.993 ... s. P. X I 20 ... 23.20 20 23.00 4 Blank previously deducted.
. ... ...
u.
...
Iodine per Tablet Mg.
0,02475 0.02613 0.02463 0,02457 0.02507 0,02638 0.02455 0.02433
TABLE V. ACCURACY OF METHOD Method Proposed
U. 9. P. X I thyroid
Weight of Sample
Per Cent of Labeled Amount of Thyroid
Iodine
95.49 96.96 95.02 94.77 96.71 97.91 94.70 93.88
Deviation from Theory
Gram
%
%
0.1000 0.1000 0.1000 0.1000 0.1000 0.1000
60.84 60,84 60.63 60.56 62.37 62.17
$0.06
+O. 06
-0.15 -0.22 +l.fiQ $1.39
639
ANALYTICAL EDITION
DECEMBER 15, 1939
tablets of all grainages of thyroid, whereas, the authors know of no other method which gives accurate and consistent results with thyroid tablets. The proposed method may be modified to make i t applicable for the determination of thyroxine in the thyroid gland and its preparations.
Acknowledgments The writers wish t o express their sincere appreciation to Edward J. Hughes and Robert M. Lingle for their assistance in the preparation of this paper.
Literature Cited Harrington, C. R., and Randall, S. S., Quart. J . Pharm., 2, 501 (1929).
Kendall, E. C., J . Biol. Chem., 43, 149 (1920). Lewis. D.. IND.ENG.CHEM..Anal. Ed.., 8., 199 (1936) Middieton, G., Analyst, 57, 603 (1932). Smith, G. F., “Ceric Sulfate”, Vol. I, 3rd ed., Columbus, Ohio, G. Frederick Smith Chemical Co., 1935. Smith, G. F., “Ortho-Phenanthroline”, Columbus, Ohio, G. Frederick Smith Chemical Co., 1935. Trevorrow, V., and Fashena, G. J., J. Biol. Chem., 110, 29 I
I
(1935); 114, 351 (1936).
A Photelometric Study of the Lead-Dithizone Svstem at 610 Millimicrons d
CHARLES L. GUETTEL, Development Laboratories, Central Scientific Co., Chicago, Ill.
I
Tu’ ADAPTIKG the Photelometer ( 5 ) to the measurement
of the lead-dithizone complex in chloroform for the quantitative determination of lead, various filters were tried with a 1-em. absorption cell and a distilled water reference solution. I n the preliminary work light filters transmitting in the vicinity of 510 m p were used, following the suggestion of Clifford and Wichmann (3). I n this spectral range the red lead complex absorbs strongly and the green dithizone transmits freely. Using the chemical procedure as outlined by the Association of Official Agricultural Chemists ( 1 ) for the mixed color method, appreciable spreads were obtained between 0 and 50 micrograms of standard lead solution. However, the small change in transmission for this lead range precluded its use as a routine method. Using the same chemical procedbe, the spread was approximately doubled when a filter having a maximum transmission a t 610 m p was substituted for the blue-green filter. The transmission curves of the lead-dithizone system as shown b y Clifford and Wichmann (2, Figure 1) indicate that such an increase in the spread is to be expected.
Experimental Using the 610 mp filter, a study was made to determine the relationship between the lead and dithizone-chloroform concentrations which would produce the greatest sensitivity for a given lead range. All reagents and apparatus were carefully purified, following the suggestions of the A. 0. A. C. ( 1 ) . Two batches of Eastman diphenylthiocarbaxone (dithizone) were purified independently and were used t o prepare two stock solutions of 30 mg. of dithixone per liter of redistilled c. P. chloroform. Solutions of 2, 4,8, 12, and 16 mg. of purified dithizone per liter of redistilled chloroform were prepared by diluting the 30-mg. per liter stock solution. Two standard lead solutions (1 ml. = 1 microgram of lead) in 1 per cent nitric acid were prepared from two individual, twice-recrystallized batches of c. P. lead nitrate, using redistilled water from a Pyrex distillation apparatus. The ammonia-cyanide mixture was prepared by pouring a quantity of redistilled ammonia equivalent to 19.1 grams of ammonia into a 500-ml. volumetric flask, adding 100 ml. of a 10 per cent potassium cyanide solution, and diluting t o the mark.
Procedure The preparation of the standard lead solutions for color development was based on the method of the A. 0. A. C. ( 1 ) . Twenty milliliters of the standard lead solution (1 ml. = 1 microgram) were pipetted into a 50-ml. volumetric flask and 1 per cent nitric acid was added to the mark. The resulting solution represented a 20-microgram lead standard in the proper condition for the color development. Standards ranging from
0 to 50 micrograms of lead were prepared by changing the quantity of the standard lead solution and diluting to the 50-ml. mark with 1 per cent nitric acid. For color development the procedure was as follows: The 50-ml. aliquot containing the standard solution was poured into a 250-ml. Pyrex separatory funnel, 10 ml. of the ammonia-cyanide mixture were added from a buret, and from another buret 15 ml. of dithiaone-chloroform solution (the concentration of dithizone depending on the lead range to be covered) were added. The separatory funnel was thoroughly shaken and the layers were allowed to separate, The chloroform layer was filtered and read immediately in the Photelometer. The water layer was discarded.
Discussion of Results Five dithizone solutions were prepared by the above procedure and the calibration curves of Figure 1 were obtained. Each curve covers the range of solutions, the colors of which vary from green to red, and all curves are asymptotic to the vertical axis. The shapes of the curves, including curve E , are characteristic of the instrument when transmission increases with concentration (4). I n general, for the filter photometer, actual transmission factors for low transmission values are usually too great. Furthermore, the vertical displacement in the substantially parallel portion of the curves corresponds to approximately 20 micrograms of lead for each change in the standard dithizone solution of 4 mg. per liter; 10 micrograms for a change of 2 mg. per liter. The curves indicate that 15 ml. of a solution containing 12 mg. of.dithizone per liter are appropriate for a lead range of 15 to 50 micrograms. For a lead range of 0 to 15 micrograms, 15 ml. of a solution containing 4 mg. of dithizone per liter are preferred. The spread between 0 and 5 micrograms of lead in scale divisions is approximately ten times as great on curve A as on curve E . However, curve A is valuable only when it is definitely known that the lead concentration of the unknown does not exceed approximately 5 micrograms. For example, using 15 ml. of a solution containing 2 mg. of dithizone per liter, the transmission of solutions containing 10, 20, 30, etc., micrograms of lead would be essentially the same, owing to the asym totic nature of the curve for transmission factors near unity. &ch a condition suggests that if the approximate amount of lead is unknown, a preliminary test should be made using 1.5 mi. of a more concentrated dithizone solution for which a calibration curve has already been prepared. If the lead is found to be extremely low, a second aliquot of the unknown may be treated with a more dilute dithizone solution, so as to utilize the greater sensitivity of the curves toward the right in Figure 1. For the range 0 to 50 micrograms of lead t w o curves are sufficient-namely, B and D. Curve B is appropriate for the
