V O L U M E 2 1 , NO. 8, A U G U S T 1 9 4 9 Because only hydrogen will diffuse with any appreciable rate ( 2 ) through the walls of the iron capsule a t this temperature, no other element will interfere with the determination and no special precautions need be taken for different samples. Originally it was thought that carbon monoxide might diffuse through a t the temperatures used. Carbon monoxide resembles hydrogen in that it would be oxidized to carbon dioxide and frozen out in the cold trap. T o test this, 0.5 ml. of iron carbonyl [Fe(CO)s], which decomposes a t 100” C. to iron and carbon monoxide, was sealed in a capsule and heated to 700’ C. in the apparatus. No indication of any increase in pressure was noted, indicating no diffusion of carbon monoxide through the walls of the iron capsule during the heating interval. Furthermore, the excess magnesium will reduce carbon monoxide to magnesium oxide and carbon in the course of an actual analysis. The nature of the blank value is still undetermined. However, it is very constant and reproducible and is equivalent to 0.015 mg. of hydrogen. All attempts to ascribe the blank value to the moisture content of the air in the capsule, or adsorbed moisture on the capsule, were without success. Nevertheless, for any group of capsulea, the blank is very constant, and need be redetermined only once a xveek during normal operations. During very humid days the blank increased somewhat, probably owing to adsorbed water on the exterior of the capsules. Perhaps degassing the capsules at a higher temperature and lower pressure than that used for the actual determination nil1 lower the blank value. The residual gas is nitrogen, which is adsorbed on the capsules after degassing when exposed to air. The identification was
1003 made with a visual spectroscope using the glow discharge which is formed as the gas is liberated. The actual amount of nitrogen is very small and is corrected for by the method of calculation. It should be possible to apply the procedure to the determination of hydrogen in higher melting metals without any changes except for a higher combustion temperature. Experiments along this line are contemplated for the future. ACKNOWLEDGMENT
The authors are indebted to Saul Dushman for informative discussions a t the inception of this project, to J. Rynasiewicz for devising the “standard” sodium hydroxide, and to W. Moak for some of the analyses included in this paper. LITERATURE CITED
(1) Bobalek, E. G., and Shrader, S.A , , IND. ENQ.CHEM.,A N A L .ED., 17, 544 (1945). (2) Dushman, Saul, “Scientific Foundations of Vacuum Technique,” New York, John Wiley & Sons, 1949. (3) Naughton, J., Trans. Am. Inst. Mech. Engrs., 162, 385 (1945). (4) Kiederl, J. B., and Niederl, V., “Micromethods of Quantitative Organic Analysis,” New York, John Wiley & Sons, 1942. ( 5 ) Norton, F. J., and Marshall, A. L., Am. Inst. Mining Met. Engrs., Tech. Pub. 1643 (1944). (6) Prescott, C. H., and Morrison, J., IND. ENG.CHEM.,A N A L .ED., 11, 230 (1939). (7) Reeve, L.,Trans. Am. Inst. M e c h . E n ~ r s . 113, , 82 (1934). RECEIVEDNovember 16, 1948. The Knolls Atomic Power Laboratory is operated b y the General Electric Research Laboratory for the Atomic Energy Commission. The n o r k reported here was carried o u t under Contract W-31-109 Eng-52
Microdetermination of Iodine in Materials with High Organic Content B. K. SHAHROKH AND R. M. CHESBRO Bio-Research Laboratories, Berkeley 2, Calif. A simple and accurate method for the determination of iodine in organic materials containing less than O.Ol’j& iodine uses the chromic acid digestion technique of Leipert followed by reduction with phosphorous acid. -4fter distillation the iodine is oxidized to iodate by means of chlorine in carbon tetrachloride solution. The iodine is then released by the addition of potassium iodide solution and measured spectrophotometrically.
To.oim
0 D E T E R M I S E iodine in compounds containing less than
iodine, . the material is digested in a mixture of chromic and sulfuric acids as described by Leipert (4). The digest is then reduced by the addition of phosphorous acid as described by Fashena and Trevarrow ( 3 , 7 ) . The liberated iodine is distilled and trapped in a special apparatus, which was developed with the cooperation of the Microchemical Specialties Company, Berkeley, Calif. After oxidation with chlorine and liberation by potassium iodide, the free iodine is determined in the Beckman quartz spectrophotometer a t a wave length of 353 mp if only a tungsten lamp is available. I f the ultraviolet attachment can be employed, it is preferable to use a wave length of 290 mp. Custer and Natelson ( 2 ) have shown that a t this wave length a 33Yo increase in sensitivity is achieved. The precision of the method a t 353 mp is such that 0.5 microgram of iodine, uncorrected for reagent blank, may be determined with an accuracy of +=0.05 microgram. At this range the spectrophotometer would give a maximum error of 10%. I t is obviously desirable to maintain the blank at a low value for most precise work (Table I).
REAGENTS
Sulfuric Acid. C.P. grade sulfuric acid contains such minute quantities of iodine that ordinarily there is no need for further purification. However, i t can be made iodine-free by adding 5 drops of 7070 phosphorous acid and boiling for 30 minutes. A 7oY0 solution of sulfuric acid is prepared from the cold concentrated sulfuric acid. Chromic Acid. If chromic acid does not contain more than 4.0 micrograms of iodine per 100 grams of solid matter, there is no need for purification. This amount of impurity which will be recovered in the blank is not sufficient to interfere with the accuracy of the results. If purification is desired the method described by Matthews, Curtis, and Brode ( 5 ) is satisfactory. A final solution of chromic acid is prepared so as to contain 70.0 grams of chromic acid per 100 ml. of water. Alkaline sulfite solution, prepared by adding 0.25 gram of sodium sulfite to 250 ml. of 0.5 A’ sodium hydroxide. 2.0 N Hydrochloric acid, prepared from concentrated C.P. hydrochloric acid which has a very low iodine content. Phosphorous acid, 7070, prepared from crystalline phosphorous acid in distilled water. Solution of chlorine in carbon tetrachloride, prepared according to the method described previously ( 6 ) , or by bubbling
ANALYTICAL CHEMISTRY
1004 Table I.
Determination of Iodine in Organic Materials
Quantity I ReUsed. covered, Gram y 0 0.22 0 0.22 0.3 0.22 0.3 0.22 Purified casein plus 0.3 0.32 0.1 y I (iodoal- 0.3 0.33 humin solution) Purified casein plus 0.3 0.54 0.3 y I (iodoal- 0.3 0.62 bumin solution)
Material Blank I Blank 11 Purified casein
Serum A
(pooled
from 9 normal
3.0 3.0
0.40 0.38
Av.
I in I ReSample, covered. y
y
%
Recovery
0.22
0
0
0
0.22
0
0
0
0.325
0.105
0.098
93
0.53
0.31
0.30
97
0.39
0.17
0.1io
100
0.37
102
0.63
95
men) Serum A plus 0.2 3.0 0.58 y I (thyroxine 3.0 0.58 0.58 0.36 solution) Serum A plus 0 3.0 0.87 7 I (thyroxin 3.0 0.89 0.88 0.66 solution) Equivalent t o 5.6 y a Average of 10 determinations. ml. of Berum. Sormal values for human serum are 5 t o 100 ml. of serum.
of iodine per 100 7 y of iodine per
Table 11. Rate of Recovery of Iodine in Distillation Apparatus Lodine Added t o Digestion Mixture, Y 0 (blank) 0.28 1.0
25 Minutes
Distillation Period 10 Minutes 7.5 Minutes Iodine Recovered
5 Minutes
Y
%
Y
70
Y
70
Y
70
0.22 0.48 1.20
100 102
0.22 0.47 1.24
100
100
0.20 0.44
0.20
102
1.18
91 93 94
91 75 75
98
0.36 0.92
chlorine gas through carbon tetrachloride until the solution assumes a bright green color. The solution is then filtered and stored in a glass-stoppered bottle; it should be resaturated with chlorine once each week. Potassium iodide solution, O.l%, prepared fresh each day just before use. It should be discarded after 1 hour. Distilled Water. Small quantities of redistilled water are needed in the last stage of the determination. Distilled water is redistilled from a potassium carbonate solution through a glass condenser. This nater is stored in a glass-stoppered wash bottle.
5 to 10 ml. of distilled water. The final volume in the flask should be around 25 ml. In order to wash the apparatus funnel B is closed and tube H n hich is connected to an aspirator is opened. The liquid in jacket L is sucked out. Water is added through A and the liquid is again emptied through H. The apparatus is then ready for the next sample.
As shown in Table 11,about 75% of the iodine is recovered after 5 minutes and almost 100% recovery is obtained after 10 minutes' distillation in this apparatus. Blank. If phosphorous acid were added to chromic acid in the distillation apparatus, there would be a violent reaction in the apparatus. In order to avoid this the following procedure should be used. In a Kjeldahl flask 3.0 ml. of chromic acid, 30 ml. of distilled nater, and 25 ml. of sulfuric acid are placed. To this are added 3.0 ml. of phosphorous acid. This quantity of phosphorous acid is not sufficient to reduce all of the chromic acid, but it will reduce enough of the acid to prevent a violent reaction. When the reaction is complete, the mixture is cooled and transferred to the distillation apparatus, 2.0 ml. of phosphorous acid are added, and the iodine is distilled as in the case of the sample. Oxidation. A piece of antibump material and 1.0 ml. of 2.0 A' hydrochloric acid are added to the Erlenmeyer flask. The flask is shaken well, 0.5 ml. of the chlorine solution is added, and the sample is boiled until less than 3.0 ml. of liquid remains in the bottom of the flask. I t is essential that the last traces of chlorine be removed from the solution and this can be accomplished only by reducing the volume of the liquid to the above-mentioned limit. However, in no case should the contents of the flask be allowed to dry. Determination. The liquid in the flask is then transferred to a test tube (100 mm. in diameter) which has previously been marked to the 4-ml. level with a diamond pencil. The level is brought to the 4-ml. line with redistilled water; 1.0 ml. of 0.1% potassium iodide solution is blown into the tube, the contents of the tube are transferred to the spectrophotometer cells, and the liberated iodine is determined in the spectrophotometer at 353 mp. Carefully matched cuvets should be employed and the transmission density should be checked against distilled water just prior to each reading of an unknown solution. The Beckman spectrophotometer is used in all determinations, and the authors' experience has been limited to this model. In addition to the directions given by the manufacturers for t h e operation of the spectrophotometer, the following precautious should be observed for greater accuracy in these determinations:
METHOD
Digestion of Sample. The material containing less than 0.3 gram of organic material is transferred uantitatively to a 500-ml. Kjeldahl flask followed by 25 ml. of 7 0 2 sulfuric acid and 3 ml. of 70y0chromic acid. As the reagents are not free from iodine, it is essential that the solutions be measured only by means of volumetric pi ets. The flask is slowly heated. When the foaming subsides feating is continued until the mixture begins to fume, by which time the contents of the flask should appear green. Bfter the flask is cool a piece of antibump material and about 50 to 75 ml. of distilled water are added to the mixture, which is then boiled until itsagain begins to fume. During the digestion, volatile acids are produced which must be removed before distillation. These acids, if resent, would pass over in the dist illation process, and woulIneutraliae the alkaline solution used for trapping the iodine. Distillation. Figure 1 is a diagram of the distillation apparatus, which employs the distillation principle used by Chaney ( 1 ) . Tube H connected to the aspirator is closed, funnel B is unclamped, and jacket C is filled with distilled water to the level of opening D. The digest is added through funnel A . The flask is a ashed twice, each time with abaut 15 ml. of distilled water, and the washings are added to the digest followed by 5.0 ml. of phosphorous acid. Funnel A and trap opening E are closed by means of pinch clamps, and 3.0 rnl. of alkaline sulfite solution are added to the trap through tube F. The water in jacket C is heated and funnel B is closed. The steam carrying the iodine is allowed to bubble through the mixture for 10 minutes. At the end of this time the flame is removed and B and A are opened. As soon as the bailing stops, the li uid in trap K is allowed to run into a 50-rnl. Erlenrnerrr flask %rough E. Thr trap is waqhpd tnire, each time with
Figure 1.
Distillation Apparatus
V O L U M E 21, NO. 8, A U G U S T 1 9 4 9 1. The cells should be checked against the control cell. Any discrepancy in the reading should be noted and corrections made in the final reading. 2. It is of the utmost importance that just before each reading the 0 mark on t,he transmission density knob be checked against the distilled water control. For this purpose the switch pointer remains on 1.0, the transmission density is set a t 0, and the shutter is opened, allowing the light to pass through the cell containing distilled water. The adjustment is made with the sensitivity control and the dark current as required in the operation of the instrument. The reading of the unknown is made immediately after this adjustment. The spectrophotometer should be calibrated against standard iodate solutions in 0.1 N hydrochloric acid. Sext, 1.0 ml. of the standard is pipetted into the 4-ml. calibrated test tubes, the volume is brought to 4.0 ml. by the addition of redistilled water, 1.0 ml. of O.lyopotassium iodide solution is
lo05 added to each tube, and the reading is made according to the above-mentioned directions. The slightly higher concentration of hydrochloric acid does not influence the results in any way. LITERATURE CITED
L., ISD. ESG. CHEM.,i l w a ~ ED., . 12, ]KO (1940). (21 Custer. J. J., and "atelson, S., ANAL.CHEM.,21, 1003 (1949). ( 3 ) Fashena. G. J., and Trevarrow, V., J . Bid. Chem., 114, 351 (1936). (4j Leipert, T., Biochem. Z.,261, 437 (1933). ( 5 ) Matthews, N. L., Curtis, G. M., and Brode, W. R., IND. ENG CHEW.,ANAL.ED.,10, 612 (1938). (6) Shahrokh, B. K., J . BWZ. Chem., 154, 517 (1914). ( 7 ) Trevarrow, V., andFashena, G . J . , I b i d . , 110, 29 ( 1 9 3 5 ) . i l l Charie), A .
Hr-i
L1VF:D . r ' l l \ '
7 . 1918.
Spectrophotometric Determination of Microquantities of Iodine JOKY J. CUSTER AND SAMUEL NATELSOK Yrooklyn College and the Jewish Hospital of Brooklyn, Brooklyn, .V. Y Procedures are described for the spectrophotometric microdetermination of iodine. Light absorption spectra are reported for iodine in water, potassium iodide solutions, benzene, toluene, alcohol, and chloroform. The high absorption peaks in the ultraviolet region for elemental iodine in toluene, benzene, and potassium iodide solutions permit these solvents to be used for the determination of microquantities of iodine with the spectrophotometer. Sensitivity is increased sixfold by converting the iodine to iodate with alkaline permanganate. The iodate reacts with iodide in the presence of acid to liberate the iodine to be determined. Iodine is transferred by extraction procedures to the benzene, toluene, or potassium iodide solutions. 4 s little as 0.2 micregram of iodine is easill determined b) these methods.
T
H E need for a method, suitable for routine use for the determination of iodine in quantities on the order of 0.2 niicrogram as found in 1 nil. of human serum, has become of ii!creasing importance. This determination is not performed in most biological laboratories because of the elaborate procedures I ) I large quantities of blood serum required for a single determiriation. The authors' purpose was to devise a procedure which could be applied with reasonable precision and accuracy to the routine determination of microquantities of iodine. Thiosulfate titration of iodine is limited to the determination d a concentration of 7.5 micrograms of iodine per ml. (65). The usc of organic solvents such as benzene, petroleum ether, chlorotorni, and carbon tetrachloride as indicators in the titration of iodine has been proposed ( 7 , 40, 4 6 ) . These procedures increase the sensitivity of the titration so that 6.0 micrograms per ml. of iodine may be detected (25). A sensitivity of approximately 2 microgram of iodine per ml. in the presence of excess of iodide ion is claimed (66). .\rsenious oxide, trivalent antimony ( 2 6 ) , sulfurous acid ( 8 ) , hydrogen sulfide ( 6 4 ) , stannous ion, and thiocyanate ( 2 1 ) have been recommended for the titration of iodine. However, none of these appears to have a greater sensitivity for the'determination of minute quantities of iodine than thiosulfate. Organic compounds such as formaldehyde ( 4 2 ) , chloral hydrate ( 4 l ) , aldoses (S), acetone ( 1 4 , 36, 49), and hydroquinone ( 5 1 ) have also been suggested for this purpose. These methods have not suggested d procedure wherein the sensitivity would be great enough to dvtermine the quantities of iodine found in 1 ml. of blood serum. Titration methods, using adsorptinn indicntors, hnwd upon
the precipitation of insoluble iodides have also been proposed (10, 11, 22, 23, 28, 29, 37, 38, 47,52). The sensitivity of these methods is less than that for the thiosulfate titration. Electrometric titration of the reaction between iodine and thiosulfate (SO) was not found practicable for routine determinations of minute quantities of iodine. The methods wherein iodine is used as a catalyst for the reaction between ceric sulfate and nitrite (18)or arsenite (9, 48, 5 0 ) are capable of determining amounts of iodine in the required range. However, these catalytic methods are delicate, and require accurate timing, careful temperature control, and special apparatus. I n this laboratory, these methods were found to be time-consuming and not uniformly successful. In view of the chromophoric character of elemental iodine itself, it was felt that a colorimetric procedure might be developed with the use of the spectrophotometer. Various colorimetric methods for the determination of inorganic iodine have been proposed (1, 12, 13, 34, 58, 43, 46). These methods use the visible portion of the spectrum in reading iodine concentration. In the visible range the extinction coefficient for iodine is not high enough to be useful for minute quantities of iodine i n mater or other solvents ( 4 6 ) . Higher peaks have been reported for elemental iodine in potassium iodide solutions in the ultraviolet ( 4 , 4 6 ) . Because the state of iodine varies with the nature of the solvent, the authors decided to investigate the absorption spectra of iodine in several solvents to see whether a higher extinction coefficient could be found. This study was extended over the entire range nf the Beckman spmtrophotometer for the purpo.e
