ANALYTICAL CHEMISTRY
798 Table I. Determination of Chromium in Catgut Sutures SO.
Catgut Sutures
of Detns.
I (Air-dried) I1 (Air-dried) I11 (Oven-dried)
Average Weight
M Q.
Average Chromium
5% 0.179
3.85 3.75 3.27
6 19 11
0.177 0.166
Average Deviation % 2.7 3.4 3.0
3Iaximum Deviation % 7 .-2 9.0 6.0
each of three concentrations (5.4, 7.2, and 9.0 micrograms) wcre determined. Two samples a t each concentration were reduced with sodium thiosulfate prior to analysis. No differences could be found in recoveries between reduced and unreduced samples. The average deviation from the amounts added was 1.7%, and the maximum deviation was 6.9%. Four samples of catgut suture, to which no chromium was added, gave the same reading as the blank. RESULTS AYD DISCUSSION
The chromium content of 1-inch strips of three sutures, prepared by the air-dried (samples I and 11)and dry-weight methods (sample 111),was determined in several separate runs. The results are indicated in Table I.
Evaluation of the results indicates that the procedure is adaptable to the measurement of minut,e quantities of chromium in catgut sutures. The average deviations from the mean are 2.7, 3.1, and 3.0% and the masimum deviations are 7.2, 9.0, and 6.070 for strips froin samples I, 11, and 111, respectively. The increase of average deviation for the samples of catgut suture over that of standard chromium solutions and recoveries of chromium added to suturcs, may result from small variations in chromicization along the length of the suture, rather than errors inherent in the procedure. ACKNOW LEDGlIENT
The authors wish to express appreciation to the Ethicon Corp., under whose sponsorship the investigation was carried out. LITERATURE CITED
(1) Kritzinger, C., and Theis, E., J . A m . Leather C h m i d s ’ d s s o c . 43, 379 (1948). (2) Lollar, R. M., Ibid., 42, 180 (1947). (3) Sandell, E. B., IWD. ENO.THEM..ASAL. ED.,8, 336 (1986). (4) Schuldiner. S., and Clardy, F.. Ihid.. 18, 728 (1946). (5) Smith, G. F., Ibid., 18, 257 (1946).
RECEIVED N a y 29, 1950
Determination of Serum Iodine Evaluation by Radioactive Tracer Technique of the Alkaline Frasion Method JOHN W. DECKER AND H E N R I E n . 4 S. HAYDEN Harper Hospital, D e t r o i t , M i c h . THE literature reports many methods for the determination of micro amounts of iodine in organic material. All procedures follow three basic steps: separation of the bound iodine from the free iodide, digestion of the bound iodine and other organic constituents of the serum, and quantitative determination of the iodine either colorimetrically or titrimetrically. However, two basically different principles underlie all these procedures: digestion in an acld medium followed by distillation or other complicated aeparations and then a quantitative determination of the iodine, and alkaline digestion followed by the quantitative determination of iodine in the presence of all the digestion products. Barker ( 1 )used a chromic acid digestion procedure, a modification of the Chaney ( 2 ) procedure, distilling the iodine into an arsenite solution, to which J+ as added a known amount of ceric ammonium sulfate. Thi5 oxidation-reduction reaction 7
(2Ce”
+
As111 yellow
-
I- 2CeIII
+ AsV)
colorless
icafalyzed by iodine as iodide. The iodine may be quantitatively estimated by measuring the time rate of change of the reaction. This relationship was first studied by Sandell and Kolthoff (7). Salter (4-6) used an alkaline digestion followed by the quantitative determination of the iodine in the presence of the fusion products, using the same ceric sulfate reaction. Salter’s (3) method for the determination of iodine in blood serum was chosen in this laboratory in preference to other procedures. This procedure used an alkaline digestion, preventing the loss of iodine by volatilization even in the presence of strong oxidizing agents. The procedure was relatively rapid, economical rn to both chemicals and apparatus, and comparatively simplei.e., free from complicated separations by distillation or preferential solvent action. At the time this procedure was adopted, work was initiated immediately to check the accuracy of the method. Radioactive
iodine (I131) was used ab a tracer element to determine quantitatively the accuracy of the procedure and also to check the reactions step by step to determine if and where the losses might occur. In order to do this the procedure was divided into four major steps and recovery was checked at each step. 1. Separation of the bound iodine from the free iodide 2. Digestion of the organic constituents in the strongly alkaline medium 3. Acidification of the alkaline fusion material befoie the colorimetric determination of iodine 4. Comparison of the amount of total iodine determined a3 such and determined as the sum of protein-bound and free iodine REAGENTS AND APPARATUS
A11 reagents for serum iodine determination were prepared as directed by Salter and Johnston ( 3 ) . Sodium hydroside, 4 N 1%and. 10% potassium nitrate, and 1.4 N sulfuric acid were used. The Geiger-Muller counter, scale of 64, self-registering type, was Model 163 manufactured by Suclear Instrument Co., Chicago, 111. A counting chamber holding the counter head a t a k e d dis tance from the sample cups was used. The cups were placed on B sliding tray which directed the cups below the counter head. The counting chamber was built by this laboratory. Stainless steel cups 1 inch (2.5 cm.) in diameter and 0.25 inch in depth were used as radioactive sample containers. Serum from either euthyroid or hyperthyroid patients waa used. The majority of these patients had previously received 50 microcuries of radioactive iodine (1131) used in a diagnostic procedure to determine the relative metabolism of the thyroid gland. In a few instances, the hyperthyroid patients had received from 4 to 6 millicuries of radioactive iodine as a therapeutic dose. In either case blood was drawn 26 hours after radioactive iodine waa given to the tracer patients and 2 to 3 hours following dosage of 4 to 6 millicuries. A saturated solution of sodium hydroside (75 grams per 100 ml. of solution) was used to ensure an alkaline medium throughout the counting. PROCEDURE
Testing Fusion Ash for Loss after Digestion. To 1 ml. of radioactive serum were added 0.5 ml. of 4 N sodium hydroside and
V O L U M E 23, NO. 5, M A Y 1 9 5 1 Table I. Saniple So.
Standard, 0.2 hll. of Serum, Counts/Min. 461 437 3647 319 8295 247 9538 188 437
799
Recovery after Ashing After Ashing, 0.2 Ml., % of Counts/.\Iin. Standard 444 96.3 427 97.7 3807 104.4 326 102.1 7942 95.7 249 100.9 952 1 99.8 101,l 190 100.0 437 BY.recorery 99 8
5
Error -3.7 -2.3 +4.4 +2.1 -4.3 to.9 -0.2 +1.1 0.0
analysis of this step triplicate samples were prepared, dried, and then compared with three standard samples of radioiodine (which are equal in amount to that which was added to the nonradioactive serum) heated to dryness only before counting. The results of this procedure are shoir-n in Table 11.
Table 11. Recovery of Radioactive Iodine Added to Seruni Standard Sample So. 1 2 3
0.1 ml. of 10% potassium nitrate in a combuEtion tube. The conibust,ion tube (10 X 1 cm. borosilicate glass) was etched with saturated sodium hydroxide previous to use. This solution was mixed and evaporated to dryness over a microburner, with care to ensure the wide distribution of solid matter in a thin film. This dark brown ash was then heated to 500" C. in a muffle furnace. The furnace was shut, off and the ash alloTyed to cool for 0.5 hour. To the ash was then added 0.5 ml. of 1.0% potassium nitrate solution, with slow washing down of the sides of the combustion tube. This dark t'urbid liquid was then evaporated again over a microburner to an expansive dry film and returned to the muffle furnace to be reheated to 500" C. The furnace was allowed to cool again for 0.5 hour and the combustion tube was removed. The ash (white) was diluted to 5.00 ml. with distilled water. Three 1.0-nil. aliquots were removed and introduced into the steel cups. One drop of saturated sodium hydroxide was added to each sample and the cups were placed beneath an infrared lamp to dry. Each cup contained 0.2 nil. of the original/ml. of serum. -40.2-ml. sample of unashed radioactive serum was introduced into each of three more steel cups plus the usual drop of saturated sodium hydroxide. These three cups contained the reference standards of original 6erum to which the 1.0-ml. aliquots from the ashed serum were compared. These cups too were dried beneath the infrared lamp. The results are noted in Table I. The average recovery after ashing was 99.8%. Each of the counts recorded in all tables was the average count of three or more like samples, with the background count subtracted in each case. Average background for this geographic location and \vith thc equipment employed was 16 counts per minute. Standard Geiger-counting procedure was used. Contamination of the counting chamber was checked first, a 3-minute background was taken ( a background count is a count of cosmic radiation for a geographical area plus the small amount of contamination near the counter), and the samples were counted from 3 to 5 minutes, depending on the concentration of the radioactive iodine. Each count was taken for 3 to 5 minutes in order to make a minimal count of approximately 1000. The results from the three aliquots were averaged after correction for background. There was no need for a self-absorption correction in any of the procedures, because every sample weighed 10 mg. per sq. cm. or less (8). The nest st,ep of the procedure to be analyzed was the separation of protein-bound iodine from the inorganic iodine. Testing Inorganic Iodine Fusion Ash for Loss during Separation. To 1.0 ml. of nonradioactive serum was added a known amount of radioactive iodine: to this solution were then added 4.0 ml. of distilled water and 0.27 ml. of 0.2 i1: acetic acid. The solution was adjusted to pI-1 6 with either dilute alkali (sodium liydroside) or acid (sulfuric acid), thoroughly mixed by agitation, and placed in a water bath at room temperature. The bath was brought to a boil, causing coagulation and flocculation and complet,ion of precipitation. The tube and contents were allowed to cool for 20 minutes a t 76" C. and were then centrifuged for 10 minutes a t 2000 r.p.m. The supernatant solution was poured into another combustion tube. The precipitate was washed three times, centrifuged with each washing, and poured into thc combustion tube containing the first washing. The washings were made alkaline with 4 drops of ammonium hydroxide (specific gravity 0.90) and slowly dried in a hot air oven a t 37' C. When dry, thifi tube was treated as when testing fusion ash for loss after digestion, except that the final ash was dissolved with 1 ml. of distilled water, and introduced int,o a steel sample cup. All radioiodine added should be found in the inorganic fraction. The usual drop of saturated sodium hydroxide was added. In the
!
(I181),
Counts/Min. 388 463 335 340 478
After Seoaration. Inurgank Iodine' Fraction (Il3l). Coimrs/Min. 394 4fi.i
,329 330 476
%
Keclamed 101,s 100.4 101.8 97.1 99 6
rr,
Errur +1.3 +0.4
f1.8 -2 -0
il 4
..in average of 100.1% was reclaimcd; the separation of inorganic iodine was adequate and therc seemed to be no absorption to or combination with protein (in vitro) hefore or after precipitztion. The protein precipitate \vas checked for radioactivitj- and the count was the same as I)acl;ground, showing that no iodine (I131)had been held by the solid protein.
Loss of Iodine. Comparison of the loss of iodine in 1 nil. of radioactive serum measured as total iodine against the loss in 1 ml. of radioactive serum calculated as the sum of the bound and free iodine entailed ashing 1-ml. samples of serum &s described above. The ash was then treated with 3 ml. of distilled water and 2 ml. of 1.4 11: sulfuric acid. The sulfuric acid reacts with the ash, liberating iodide which was to catalyze the arsenious acidceric ammonium sulfate reaction. The ash dissolved in the dilut,e solution of iodine-free sulfuric acid with some effervescence. The effervesrenre of reaction is kept, well under control by gently agitating the combustion tube until the solution is complete. Three 1-ml. aliquots were transferred to steel cups, made alkaline, and dried as previously noted. Three 0.2-nil. samples of the same untreated radioactive serum were prepared and dried an previously explained. The tKo sets of sanipleR were compared for loss during acidification of the iodine. The results are shown in Table 111.
Table 111. Recovery of Total Iodine after Acidification Sample No.
Standard, 0.2 MI., Counts/Min.
After Acidification 0.2 M1. Cou&/Min:
%
Recovery
%
Error
A r . ravovery 8 3 . 2
For checking the loss during acidification of the protein-t)ound iodine and iodide fraction, the combined counts from the two fractions separated before fusion wcre compared to 0.2 ml. of radioactive serum. The 1.0-ml. sample of radioactive aerum was separated as in testing inorganic fusion ash for loss during separation and the organic curd and washings (iodide) were treated as in testing fusion ash for loss after digestion. The resulting fusion ash was dissolved with 3.0 ml. of distilled water and 2.0 ml. of 1.4 N sulfuric acid and aliquots were taken from each tube and treated for the counting procedure. Therefore, the count from 0.2 ml. of radioactive serum \vas the standard and the count from 1.0 ml. of protein-bound iodine plus 1.0 nil. of the iodide fraction was compared to the standard. The 1.0 ml. of protein-bound iodine plus the 1.0 ml. of iodide was equal to 0.2 ml. of the original 1.0 ml. of serum. The results from this analysis are presented in Table IV. Comparison of the results recorded in Table I11 (average recovery of total iodine = 83.2y0) with those in Table IV (average recovery of total iodine, protein-bound iodine PIUS iodide =
aoo
ANALYTICAL CHEMISTRY
99.3%) shows that a more complete recovery of tot.al iodine is obtained by summation of the bound and free iodine determined separately.
Table IV. Recovery of Total Iodine after Separation and Fusion Sample No.
DISCUSSION
The experimental evidence found by the procedures described in this paper indicate that losses occurring during the alkaline fusion procedure were negligible. The experimental evidence indicates that acidification of smaller amounts of ashed serum per unit volume of solution resulted in a smaller loss. This was proved by the fact that the total iodine determined in 1 nil. of serum was of the order of 82.3% of the original serum, or of the total iodine calculated as the sum of the protein-bound iodine contained in 1 ml. of serum and the inorganic iodine in the same 1 i d . of serum. Hence, a dilution of 100% gave a 99.3% recovery. Thus it is necessary onlj- to determine protein-bound and free iodine, oniitt.ing the step of determining t,otal iodine ae such. From 100 cases t,he total iodine deterniined on 0.5 ml. of serum chemically varied in the order of 1 0 . 2 microgram from the total iodine calculated by the t x o sepnrat>efractions.
Protein-Bound Iodine and Iodide Counts
Standard Counts
.4v. recovery
7%
Recovery
%
Error
99.3
fraction by separation before digestion (Table IV average recovery, 99.3%). There is a large and variable loss upon acidification of the total iodine sample following digestion (Table I11 average recovery, 83.2%). This may be avoided by the modification of the method presented in this paper. LITERATURE CITED (1) Barkey, S. B., J . Bid. Chem., 173, 715 (1948).
COSCLUSIONS
Salter’s method for blood Serum iodine gives both precision and accuracy, if total iodine IS calculated as the sum of two fractions, inorganic and orga:iic. The biologically important proteinbound iodine is shown to be rletelmined accurately. The precipitation of protein-bound iodine is adequate as noted in Table I1 (average recovery 100.1%). The alkaline digestion procedure gives an average recovery of 99.8% (see Table I). An adequate recovery of total iodine present is obtained by combining the results of the protein-bound fraction and iodide
(2) Chaney, A. L., INDENQ.CHEM.,ANAL.ED., 12, 179-81 (1940). (3) Salter, W.T., and Johnston, RlacA. W., J . Clin. Endocrinol., 8, 911-33 (November 1948). (4) Saltei, W.T., Johnston, Macii. W., and Gemmel, J., Symposium on Radioiodine, Brookhaven National Laboratories, pp. 24-34, July 1948. ( 5 ) Salter, W T., Karandikar, G., and Block, P., J . Clin.Endocrinol., 9, 1080-98 (Sovember 1949). (6) Salter, W. T., and hfcKay. C. A., J . Biol. Chem., 114, 495 (1936). (7) Sandell, E. B , and Kolthoff, I. SI.,Mik~ochim.Acta, 1, 9 (1937). (8) Seaborg, G. T., Jaffey, A. H., Kohman, T. P., and Crawford,
J. A.. “Manual on the Measurement of Radioactivity,” U. S. Atomic Energy Commission, MDDC-388 (1944). RECEIVED April 14,19.50.
Conversion of Pfund Gage Reading to Dry Film Thickness M. H. SWITZER Continental Can Co., Inc., Chicago, I l l . H E Pfund gage ( 2 ) has for many years been employed for Tmeasuring the wet thickness c s of applied organic coating films. Briefly, this instrument is a plano-convex lens of known radius of curvature, mounted so that its conves surface can be pressed reproducibly int,o the \vet coating film down to the substrate; the diameter of the paint spot thus produced on the convex surface of the lens is employed as a nieaiure of the thickness of the film. The author has frequentl?. needed to determine the thickness of dried coating film. where direct measurement is inconvenient. In many instances, direct nieawrement of the thickness of the dried film is either very difficult or impossible. because of the nature of the article t.o which the coating is applied. On the other hand, very few instances have been found in which the Pfund gage could not be employed to obtain an estimate of wet film thickness; later touching up of the emall marks left in the finish is ordinarily a simple procedure. .\ccordingly, an equation has been developed relating the Pfund gage reading to the resulting dry film thickness and x nomogram ronstructed for the solution of this equation throughout the useful arguments of its factors. The equation is developed as follows: Let 7’ = thickness of wet film, millinieters L = diameter of spot on Pfund film thickness gage, millimeters r = radius of curvature of lens of Pfund gage = 250 rnm. Then
Let TV = weight in milligrams of wet coating film on any selected area, A A = selected area, square inches G = weight per gallon of coating material, pounds as prepared for application M = mm. per inch = 25.40005 n = cubic inches per gallon = 231 q = mm. per pound = 453592.4277 Then (Thickness of wet paint in inches) = (weight of wet film per sq. inch) (weight of 1 cu. inch of coating) Hence,
Combining Equations 1and 2, _La= -
W AG
7
LZAG X
9 E
16r
mn
and
W Let S
=
(3)
= per cent solids of the coating material divided by
10O-i.e., weight of a sample of the coating material after drying in accordance with a schedule
