Table 111.
Wt. % Glycol 0
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 pure 0
10.00 20.00 30.00 40.00 50.00 60,OO 70.00 80.00 90.00 pure 0
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 Pure
Table IV.
Smoothed Data
Abs. Density, G./MI. R.I., nn at 25" C. At 20" C. -4t 25" C. Triethylene Glycol 0.99707( 4 ) 1.3330 1.0117 1.3449 I.0271 1.3572 1.0431 1.3705 1,0591 1.3834 1.0745 1.3970 1.3956 1 ,0884 1.4103 1.4086 1.4226 1.4210 1.1001 1.4345 1.1092 1.4330 1.4453 1,1158 I.4437 1.4558 1,4541 1.1196 Dipropylene Glycol 0.99707( 4 ) 1.3330 1.0046 1.3452 1.0131 1.3578 1.0216 1.3709 1.0288 1 3836 1.0338 1.3962 1.0359 1.4071 1.0354 1.4171 1.0322 1.4263 1 ,0269 1.4343 1.0165 1.4407
1,3325 1.3446 1.3569 1.3698 1.3822 1,3944 I ,4054 1.4155 1.4246 1,4326 1.4389
Hexylene Glycol 0.99707( 4 ) 1.3330 0.9967 1 ,3458 0.9972 1 ,3589 0.9962 1.3717 0.9918 1.3833 0.9851 1.3937 0.9764 1.4032 0.9660 1.4118 1.4190 0,9539 1.4248 0.9389 1.4275 0.9181
1.4013 1.4100 1.4171 1.4228 1.4257
Applicability of Eykman Equation
Triethylene glycol Dipropylene glycol Hexylene glycol
Value At 20"C. 0,5369 0.5728 0.6156
of C,
At 25OC. 0.5369 0.5727 0.6160
icals Co., a division of Union Carbide Gorp., for contributing the samples of glycols used in this work. This project was supported by a grant from the Engineering Experiment Station of the University of Rhode Island. LITERATURE CITED
Curme, G. O., Johnston, F., "Glycols," ACS Monograph 114, Reinhold, Sew Tork, 1953. Fogg, E. T., Hixson, il. I., Thompson, A. R., ANAL. CHEM. 27, 1609 (1955). (3) Kurtz, S. S., Jr., Amon, S., Sankin, h., Znd. Eng. Chem. 42, 174 (1950). (4) Lange, K.A, "Handbook of Chemistry," 9th ed., Handbook Publ., Sandusky, Ohio, 1956. MacBeth, G., Thompson, A. R., ANAL.CHEM.23, 618 (1951). Zbid., 24, 1066 (1952). Kard, A. L.,Kurtz, S. S., Jr., IND. ENG.CHEM.,AXAL. ED. 1 0 , 573 (1938). RECEIVEDfor review April 18, 1957. Accepted June 15, 1957.
Chloric Acid Method for Determining Protein-Bound Iodine by Use of Iodine-131 JESSE F. GOODWIN, RICHARD B. HAHN, and A. J. BOYLE Department of Chemistry, Wayne State University, Detroit 2, Mich.
b A study of the loss of iodine-1 31 during the digestion of samples with chloric acid has shown that addition of chromate to the sample is unnecessary. Protein-bound iodine can b e separated from inorganic iodide by using either perchloric or trichloroacetic acid as a protein precipitant.
for the quantitative determination of protein-bound iodine have been reviewed by several workers ( 2 , 4,6). This study is concerned with losses of iodine related to modifications of the chloric acid method (1, 3, 5 ) for digesting samples. Chloric acid digestion makes it possible t o prepare a sample for analysis in a ETHODS
single container; this obviates losses of iodine during transfer. It has been reported that chloric acid mixed with small amounts of sodium chromate is the most satisfactory reagent for sample digestion because of its powerful oxidizing action a t relatively low temperatures. The effect of chromate in this digestion mixture has not been sufficiently investigated. Iodine-131 has been used in this study t o determine the usefulness of chromate in preventing iodine losses during chloric acid digestion. STANDARDIZATIONS OF TRACER SOLUTIONS
A tracer solution of iodine-131 was oxidized from sodium iodide to iodat,e
by boiling it nearly to dryness with 5 ml. of chloric acid and 10 mg. of sodium chromate. This preparation was cooled, the residue mas dissolved in 5 to 10 nil. of distilled water, and the solution volume was adjusted t o 25 ml. A welltype scintillation counter was used t o obtain 5-minute counts on 2-ml. aliquots of this solution. An average of 4900 counts per minute with a probable error of 1 8 8 counts per minute (1 I .76%) for the individual samples was found by counting six aliquots (Table I). Corrections were made for background count.
A more concentrated solution of iodine-131 iodate prepared and standardized. Six aliquots of this s o h tion gave an average of 21,878 counts VOL. 29, NO. 1 1 , NOVEMBER 1957
1681
per minute. The piobablc error for a single counting of this solution was calculated to be *309 counts per minute ( 1 1.41%;). The error resulting from taking successive counts of the samples a t 2-hour intervals was found to be & 1.54%. Several preparations of iodine-131 iodate of varying radioactive strengths were used in this investigation. RECOVERY
OF IODINE-131
AS IODATE
After Chloric Acid Digestion. T h e loss of iodine when standard preparations of iodate are digested Ivith chloric acid a n d chromate was determined by this experiment. (Samples in this a n d ensuing experiments were digested in 180-ml. electrolytic beakers.) Standards containing 0.1, 0.15, and
0.2 y of nonradioactive iodate were prepared in quadruplicate. To each mas added a 2-ml. aliquot of iodine-131 iodate, 1 ml. of 5% sodium chromate solution, and 25 ml. of 20% chloric acid. These preparations were placed on a hot plate at low temperature (120' C.) and allowed to evaporate to approximately 0.5 ml. Four 2-ml. aliquots of iodine-131 iodate which were not subjected to the digestion procedure served as controls against which the final activity of the digested samples was measured. The results shown in Table I1 indicate that the digestion procedure is responsible for approximately a 59T0 loss in activity. (Throughout this
Table 111. Recovery of Iodine-131 Iodate from Chloric Acid Digestion without Added Chromate
IO3-, Addedy Table 1.
Standardization of Tracer Solutions
Counts Deviaper tions, Sample Minute X XZ 1 4,853 137 18,769 2 4,910 80 6,400 3 5,063 73 5,329 4 5,115 125 15,625 5 4,860 130 16,900 6 5,140 150 22,500 Av. 4,990 116 Probable error for single measurement. 0 . 6 7 4 5 4 s where n is the number of observations. Probable error. 4900 f 88 yo probable error for single measurement. f1.76 Table II. Recovery of Iodine-131 Iodate after Chloric Acid Digestion
Added % IO3-, C. P. M. Recovery 0.20 19,782 91.61 0.20 20,685 95.78 20 835 96.48 0.20 0.20 20 ;559 95.21 0.15 21,045 97.46 0.15 20,811 96.37 0.15 20,361 94.29 0.15 21,015 97.32 98.24 0.10 21,215 18,555 85.93 0.10 19,089 88.40 0.10 0.10 21,197 98.16 Av. 20,429 94.60 Probable error for single measurement. 20,429 f 590 yoprobable error. 12.89 Controls c. P. nr. 1 21,412 2 21,837 21,401 3 21,730 4 21,594 Av * Probable error for single measurement. 21,595 & 389 % probable error. f 1 . 8 0 I
1682
ANALYTICAL CHEMISTRY
study controls of iodine-131 iodate were not digested.) A similar series of standards was digested without the addition of chromate, to determine whether iodine loss would occur. The results shown in Table I11 indicate that chromate is unnecessary to protect from iodine loss during digestion. Effect of Added Chromate upon Recovery from Blood Serum. Samples of iodine-131 iodate a n d nonradioactive iodate were placed in electrolytic beakers for digestion with chloric acid as described. One milliliter of pooled human blood serum was added to each beaker. I n order to test the effect of added chromate upon iodine recovery, the group of samples was divided into two corresponding series. Chromate was added to one series and omitted from the other. All samples were digested by boiling them to incipient dryness. Table IV shows that little or no advantage is obtained by addition of chromate to the digestion mixture. Digestion Continued to Complete Dryness. Duplicate series of standa r d preparations of iodine-131 a n d nonradioactive iodate were digested with chloric acid. Chromate n-as omitted from one series. These preparations were digested t o complete dryness. Digestion was taken t o be complete when acid fumes ceased to evolve even when the temperature of the hot plate was increased. The presence of chromate tends to give somewhat more consistent results or less probable error. It is apparent that evaporating
Recovery %
C. P. RI.
0.20 19.709 99.78 0.20 19;437 98.40 0.15 19,096 96.67 19,330 97.85 0.15 0.15 19,056 96.47 19,443 98.38 0.15 19,663 99.54 0.10 0.10 19 ;458 98.51 Av. 19,399 98 18 Probable error for single measurement. 19,399 i 158 probable error. 10.81 Standard Controls 2 Av .
Table 1V.
c. P. &I. 19,741 19,766 19,753
1
Recovery of Iodine-131 Iodate in Blood Serum after Chloric Acid Digestion with and without Added Chromate
Without Chromate Added IO$-, Y 0.20 0.20 0.20 0.20 0.15 n- . 1.5 -_ 0.10 0.10 Av. Probable error for single measurement % probable error Controls 1
2
3
4 Av . Probable error for single measurement % probable error
C.p.m. 5,828 5,671
recovery 97.72 95.09
5 ;676 5.721 -.--5,645 5,738 5,713
95:i7 95.93 94.65 96.21 95.79
...
5,713 & 82 i 1.44 C.P.M. 5,862 6,160 5,869 5,964 5,964 5,964 f 65 f 1.09
With Chromate
%
C.p.m. 36,473 35,593 35,760 36,465 36,438 36.626
...
3 6 ;703
36,550 36,326
Controls 1
2 3 4
%
recovery 102.18 99.71 100.19 102.16 102.07 102.60 102.93 102.40 101.76
36,326 =k 197 & 0.54 C.P.M. 35,881 35,637 35,152 36,116 35,696 35,696 =k 320 f 0.90
form of potassium iodide was prepared. Four identical samples were made u p by mixing 3-ml. portions of this standard and 2-ml. portions of pooled human blood serum in 40-ml. round-bottomed centrifuge tubes. Initial counts &-ere taken. Twenty-five milliliters of 15% trichloroacetic acid reagent were mixed with the contents of each tube, t o precipitate the proteins. The protein precipitates were packed by centrifuging the samples for 10 minutes at 2000 r.p.m. The supernatant liquid v a s decanted and a count was obtained on each precipitate. The precipitates were then washed by suspending them in 25-ml. portions of trichloroacetic acid reagent, centrifuging them, and decanting the supernatant liquid. Counts were taken on the washed mecbitates. The results of this experimeit are' shown in Table VII. This experiment was repeated on duplicate samples using 1.5N perchloric acid instead of trichloroacetic acid
to complete dryness invites inaccuracy (Table V). Effect of Added Chromate When Digestion Continued t o Complete Dryness. Duplicate series of standa r d preparations of iodine-131 iodate, nonradioactive iodate, a n d 1 ml. of pooled human blood serum were digested t o complete dryness with chloric acid. Chromate was omitted from one series. The effect of chromate upon iodine recovery after digestion to dryness is shown in Table VI. It is apparent that whether chromate is present or not, the iodine is largely lost when serum samples are taken to complete dryness; the presence of organic components may be responsible for this gross loss. Separation of Protein-Bound from Inorganic Iodine by Protein Precipitants. A standard solution containing a quantity of NaI131 and 1 y per ml. of nonradioactive iodine in t h e
Table V.
Recovery of Standard Iodine- 13 1 Iodate When Digestion Is Continued to Complete Dryness Wit.hout Chromate With Chromate
Added Y
103-,
0.20 0.20 0.15 0.15 0.10 0.10 0.10
0.10
-4v. Probable error for single measurement yo probable error
C.p.m. 20,969 20,783 27,608 27,371 21 ;381 26,046 21,201 27,371 24,091
70
recovery 79.53 78.82 104.71 103.81 81.10
98.79 80.41 103.81 91.35
24,091 5 2,194 f 9.11 Controls NO. 1
2 3 4
5 6
Av. Probable error for single measurement yoprobable error
C.p.m.
24,408 24,040 25,005 25.356 23 444 22,532 24,508 22,181 23; 934
23,934 =t765 rt 3.20 C.P.M. 26,968 26,174 26,016 25,970 26,149 26,917 26,365
C.p.m.
y
26,365 f 262
* 1.00
1
2 3 4
Controls 1
2
3 Av.
C.p.m. 26,968 25,970 26.917 26; 618
% -
recovery
C.p.m.
Table VIII. Separation of Inorganic Iodine from Protein-Bound Iodine b y Precipitation with Perchloric Acid YO.
CoprecipitaC. P. 11. tion Initial Counts A1 ... 7910 7372 A2 ... Counts after Initial Precipitation -41 496 6.49 2 557 7.29 Av. 6.88 Counts after Washing AI 45 0.59 A2 18 0.24
Sample
(Table VIII). It is apparent that either trichloroacetic or perchloric acid is satisfactory for the separation of protein-bound iodine from the free iodide present in serum and that one washing is sufficient for clinical laboratory purposes.
Recovery of Iodine-1 31 Iodate in Serum after Digestion to Complete Dryness with and without Added Chromate Added Without Chromate With Chromate ~OS-,
Counts after Initial Precipitation 1 416 4.16 '7 326 3 04 396 3.69 3 418 3.90 4 AV. 396 3.70 Counts after Washing 1 82 0.76 2 6 0.06 3 36 0.34 4 15 0.14 iiv. 35 0.33
70
recovery 92.57 91.18 94.84 96.17 88.92 85.46 92.95 84.13 90.73
Table VI.
0.2
Table VII. Separation of Inorganic Iodine from Protein-Bound Iodine b y Precipitation with Trichloroacetic Acid So Inorg. Iodide CoprecipiSample C. P. M. tated Initial Counts 1 10.844 2 10 767 ... 3 10,418 ... 4 10,865 ... 4V. 10,723 ...
%
recovery
DISCUSSION
It is evident from this study that chromate nred not be used when serum samples are digested by the chloric acid method. If digestion is continued to complete dryness, the iodine in a sample of serum is almost wholly lost even when chromate is present. Recoveries of iodine are not significantly affected by the presence or absence of chromate when digestion is stopped before complete dryness has been reached. !A7hen samples that contain no organic matter are digested VOL. 29, NO. 11, NOVEMBER 1957
1683
to complete dryness, chromate helps to prevent iodine loss.
(2) Moran, J. J., ANAL. CHEW 24, 378
(1952). (3) O'Neal, L. W., Simms, E. S., Am. J . Clin. Pathol. 23, 493 (1953). ( 4 ) Van ZYlt A.7 8. African J . Sci. 16, 95 (1951). (5) Zak, B., Willard, H. H., Myers, G.
LITERATURE CITED
(1) Leffler, H.H., Am. J . Clin. Pathol. 24, 483 (1954).
B., Bogle, A. J., ANAL. CHEW 24, 1345 (1952).
RECEIVED for revieJv February 2, 1957. -4ccepted July 25, 1957. Supported by a grant from the Michigan Heart dssociation.
Titrimetric Determination of Sdfamic Acid C. L. WHITMAN Research and Development Department,
U. S.
b A method has been developed for the titrimetric determination of small amounts of sulfamic or nitrous acid in the presence of nitric acid. The procedure involves the reaction of sulfamic acid with sodium nitrite, oxidation of excess nitrite with cerium(lV) ammonium sulfate, and titration of excess cerium(lV) ion with iron(l1) ammonium sulfate. From the relation between the amount of iron(l1) ion required for the sample, and that required for two different blanks, it can b e determined whether nitrous or sulfamic acid is present, and the amount of either can b e calculated.
A
was sought for the determination of small amounts of sulfamic acid. The expected concentration was 0 to 10 ml. of 0.01M sulfamic acid per 100 ml. of solution. If no sulfamic acid were present, free nitrous acid would be present and this would have to be determined instead. Two gasometric methods for the determination of sulfamic acid have been described. Meuwesen and hlerkel (4) treated sulfamate solutions with dilute sulfuric acid, added sodium nitrite, and measured the nitrogen evolved after absorption of any nitric oxide in alkaline permanganate solution. The method devised by Carson (3) involves the reaction of the sulfamic acid with sodium nitrite, absorption of nitric oxide in bromine and water, and measurement of the volume of nitrogen. The gravimetric method of Baumgarten and Krurnmacher (1) is based on the precipitation, as barium sulfate, of any sulfates present in the sample, reaction of the sulfamic acid with sodium nitrite in acid solution and a second precipitation as barium sulfate, of original sulfate plus that from the oxidization of sulfamic acid. A titrimetric method developed by Bowler and Arnold ( 2 ) uses the reaction of sulfamic acid with nitrous acid and detection of excess nitrite ion with starch-iodide indicator. The method developed in this work METHOD
1684
ANALYTICAL CHEMISTRY
Naval Powder Factory, Indian Head,
Md.
was a titrimetric determination of small amounts of sulfamic or nitrous acid in the presence of nitric acid. This method is valid, even though it is not known which of the two constituents is present. However, a qualitative test for nitrous acid may be made with starch-iodide paper. Among the methods tried &-as the addition of excess nitrite ion and determination of the unreacted nitrite colorimetrically with 1-naphthylamine m d sulfanilic acid. The color was slow in forming, a precipitate was formed a t high concentrations, and the accuracy was not high enough. The gasometric method had the drawbacks that the amount of gas released might be extremely small, and the presence or absence of nitrites was not determined. Attempts to break dona sulfamic acid with sodium hydroxide were not successful. The method which gave the most satisfactory results involves the following series of reactions: NHzS03H
+ T\TaN02
+ NaHSOh
+ XZ+
Hz0 (1)
+ S O 2 - - + KO3- + 2 Cef3 Ce+4 + Fe+2+ Ce+3 + Fe+3
2 Ce+4
(2)
(3)
To allow for the fact that in a given solution either nitrites or sulfamic acid may be present, two blanks are used. Blank 1 indicates the amount of cerium(1V) used and also furnishes data for the standardization of the iron(I1) solution. Blank 2 shows the amount of nitrite used. The relationship between the volume of the blank and the volume of standard iron(I1) solution indicates which of these two constituents is present. As indicated in the equations, the method is based on the reaction of sulfamic acid (if present) with acidified sodium nitrite. Nitrite originally present if sulfamic acid is absent, plus any unreacted nitrite that is added later, is oxidiEed with cerium(1V) and the excess cerium(1V) titrated with standard iron(I1) using o-phenanthroline iron(I1) sulfate as the indicator.
REAGENTS
Iron(I1) ammonium sulfate solution is prepared by adding 8 grams of the hexahydrate to 20 ml. of sulfuric acid (1 to 1 by volume) and diluting to 1 liter. The resulting solution is a p proximately 0.02N. Cerium(1V) sulfate solution. The 0.02N solution is made by taking 13 grams of the dihydrate, adding 40 ml. of sulfuric acid (1 to 1 by volume), and diluting to 1 liter. Sodium nitrite, 0.01M. Exactly 0.6901 gram of dried sodium nitrite are dissolved in water and the solution is diluted up to the mark in a 1-liter volumetric flask. o-Phenanthroline iron(I1) sulfate indicator, 0.02511f. Fisher P-69 or equivalent is satisfactory. STANDARDIZATION
The 0.02N cerium(1V) sulfate solution is standardized against a standard solution of sodium thiosulfate. A 25ml. sample of cerium(1V) sulfate solution is pipetted into an iodine flask and 5 ml. of 15% potassium iodide and 10 ml. of hydrochloric acid (1 to 1 by volume) are added. After standing for 5 minutes, the solution is titrated with sodium thiosulfate to a starch end point. The normality, N , of the iron(I1) solution is obtained from blank 1 as
where VI is the milliliters of cerium(1V) solution, and N I its normality, while V z is the milliliters of iron(I1) solution. The normality of the sodium nitrite is not used in the calculations and therefore it is not necessary to have an exact value for its normality. If desired, its concentration may be calculated from one of the values of the blank. PROCEDURE
A 100-ml. sample of the material to be analyzed is placed in a 500-ml. iodine flask and 20 ml. of 1 to 1 sulfuric acid (by volume) are added. A known volume of 0.01M sodium nitrite, in excess of the amount required for the
