797
V O L U M E 23, NO. 5, M A Y 1 9 5 1 tube spacer wound on a spool, to the inlet of the comparison tube, and from thence to the receiver, U. Thus, refractive index gradients across a volume interval equal to the spacer are measured. As long as the concentration of substance in the effluent is constant, the refractive index gradient is zero. When a zone of substance arrives in the cuvette, the refractive index rises to a new level, and this is then observed as a spike. Thus, rather than a stepwise rise in refractive index, one obtains a series of maxima rising from the base line, corresponding to the steps of the ordinary displacement diagram. In the discussion here, displacement separation has been used as an example. The apparatus is well adapted also to elution, frontal, and carrier displacement separation. ACKNOWLEDGMEhT
Opportunity is taken to commend the workmanship of D. Milton Kvanbeck of Minneapolii, Minn., who constructed the custom-built model discussed in this paper. This work ww
supported by a granbin-aid from the Williams-Waterman Fund of the Research Gorp. LITERATURE CITED
(1) (2) (3) (4)
Claesson, S., Arkiv Kemi Mineral. Geol., 23A, 1 (1946). Ibid., 26A,24 (1949). Hagdahl, L., Acta Chem. Scaad., 2, 574 (1948). Hagdahl, L., and Holman, R. T., J . Am. Chem. Soc., 72, 701
(1950). (5) Holman, R. T., AN.4L. C H E Y . , 22, 832 (1950).
(6) Holman, R. T., and Hagdahl, L., Arch. Biochm., 17,301 (1945). (7) Holman, R. T., and Hagdahl, L., J . B i d . C h m . , 182,421 (1950). (8) Moring-Claesson, I., Biochim. et Biophys. Acta, 2,389 (1948). (9) Tiselius, A,, Arkiv Kemi Mineral. Geol., 16A, 18 (1943). (10) Tiselius, A., “The Svedberg,” Stockholm,4lmquistandT~iksells, 1944. (11) Tiselius, A., and Claesson, S., Arkiv Kemi Mineral. Geol., 15B,18 (1942). (12) Tiselius,A , , and Hahn, L., KoZloid Z . , 105,177 (1943). RECEIVEDAugust 28, 19.50. Presented before the Division of $nalyticsl Chemistry at the 117th hfeeting of the AZ~IERICAN Cx~narc.4~ SOCIETY. Houston. Tex.
Microdetermination of Chromium in Catgut Sutures HERBERT Y, WEISS, \-ISTON E. SILER,
AYD
PETER R . BUECHLER
Department of Surgery, t-niversily of Cincinnati, Cincinnati, Ohio
TITRIMETRIC method for the determination of chromium
A in catgut mtures, using 1-to %gram samples, has been re-
ported ( 5 ) . However, studies in progress a t this laboratory involve the determination of chromium in small strips of catgut suture, weighing 3 to 4 mg. As the chroniium content of these sutures ranges between 0.15 and 0.2575, a more senfiitive method of analysis is required. The high sensitivity of the diphenylcarbazide method for determining chromium (3) has been adapted to the determination of chromium in leather ( 2 ) . The procedure described is not within the working range for the minute quantities of chromium to be determined in catgut sutures. AIodifications of this procedure have, however, afforded the accurate determination of chromium in 3- to 4-nig. samples of catgut suture. The method is simple, rapid, and should he generally applicable to the determination of minute amounts of chromium in biological materials-e.g., in the stratoassessographic analysis of chrome-tanned leather ( 1 ) . Iron, vanadium, molybdenum, and mercury interfere with color development in the diphenylcarbazide method. Of these, iron is the only interfering metal that may possibly be encountered in catgut sutures. (Phenyl mercuric benzoate is added to certain tubing fluids. However, the amount employed is insufficient to produce interference in the diphenylcarbazide reaction.) The addition of phosphoric acid, to complex the iron possibly present, effectively minimizes this interference ( 2 ) . A 108s of chromium in the perchloric acid oxidation, resulting from the formation of ehromyl chloride, may occur ( 4 ) . This is obviated by adjusting the temperature below the boiling point of constant boiling perchloric acid, and yet maintaining a sufficiently high temperature to permit complete conversion of chromium t o the hexavalent state. PREPARATION OF SAMPLE
The sutures, following removal from the tubes, were cut into 1-inch (2.5cm.) strips, allowed to come to equilibrium in a room which remained relatively constant with regard to humidity and temperature, and then weighed, For determining absolute values of chromium content, 1-inch strips of euture were transferred to micro weighing bottles, oven-dried under vacuum a t 70” C. for 18 hours, and weighed. REAGENTS
Buffer solution, pH 1.6. Take 250 ml. of 0.2.Y potassium chloride (14.9 gramF per liter), mix with 150 nil. of 0.2 -Irhydro.
chloric acid (17.1 grams of conctntrated hydrochloric acid per liter), and dilute to 1 liter. Diphenylcarbazide, 0.25% in 1 to 1 acetone-water. This solution should be prepared daily and kept in the dark while not in use. EXPERI?vlENTAL PROCEDURE
The sample is transferred to a 12 X 75 mm. test tube. Two drops of concentrated sulfuric acid and 4 drops of concentrated nitric acid are added, and the mixture is boiled gentlyon a hot plate until carbonization occurs. The sample is cooled, and one drop of hydrogen peroxide (3070),isadded. The mixture is heated again to charring, removed from the hot plate, and cooled, and the treatment with a drop of hydrogen peroxide is repeated. Usually 3 drops of hydrogen peroxide are required to effect complete digestion of the sample in this manner. Following clearing, the sample is allowed to remain on the hot plate for 0.5 hour to remove traces of unreacted hydrogen peroxide. .-ifter cooling, 2 drops of constant boiling perchloric acid (70 t o 72%) are added to the digestion tube. The test tube iz inserted about. 15 mm. below surface of a constant temperature bath adjusted at 200” * 2 C. and oxidized for 15 minutes. After oxidation, the tube is immediately inserted into a cold water bath and diluted with distilled water to a volume of approximately 3 ml. The solution is transferred into a 25-ml. volumetric flask. About 10 ml. of water are used for the transfer. The volumetric flask is placed on a hot plate and the solution is boiled for several minutes to remove liberated chlorine. After cooling, one drop of concentrated phos horic acid and 10 ml. of the buffer solution are added. One milliher of diphenylcarbazide is transferred to the flask, water is added to the mark, and the solution is shaken. The color is allowed to develop for 1.5 minutes. Readings are made in an Evelyn colorimeter using a S o . 540 Evelyn filter.
tp
PREPARATION OF ST.AYDARD GRAPH
Known samples of hexavalent chromium a t five concentrations, ranging from 3.6 to 10.8 micrograms, were transferred to the digestion tubes, reduced with an excess of sodium thiosulfate, digested, and oxidized in the manner described. Six samples at each concentration were determined. The color developed was read against a blank solution treated similarly. Per cent transmittance for the concentrations indicated ranged from 25 to 65. The optical density of the mean reading a t each concentration was plotted; Beer’s law was followed. The average deviation from the mean values of opt’ical density at the particular concentrations was 1.i’70 and the maximum deviation from the mean (at 3.6 micropr:tnis) was 6.2% RECOVERY EXPERIMEIITS
Solutions of potassium dichromate were added to 1-inch (2.5cm.) strips of ordinary nonchromicized catgut. Four samples a t
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)
6 19 11
Average Weight
M Q.
Average Chromium
5% 0.179
3.85 3.75 3.27
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
