ANALYTICAL CHEMISTRY
378 the determination of ethyleiiediaminetetraacetic acid in urine is of some importance, The following experiment in duplicate was made with this in mind.
containing known amounts of Sequestrene NA3 could be used instead of decolorizing with Nuchar. ACKNOWLEDGMENT
An 8-mg. Sequestrene NA3 test solution was prepared, using as the sample 50 ml. of urine diluted with 50 ml. of distilled water. To the sample were added 15 ml. of nickel solution; after 10 minutes 5 ml. of C.P. ammonia (28%) and 15.5 ml. of dimethylglyoxime solution were added. After 2 minutes the sample was stirred with 3 grams of Nuchar C115 (activated carbon from Industrial Chemical Sales, Kew York, N. Y.) as a decolorizing agent. The solution was filtered after 5 minutes. The filtrate had a very pale yellow color. A further addition of 4 grams of Nuchar C115 was made and the sample again filtered. The filtrate was now practically colorless. To 50 ml. of filtrate were added 2.5 ml. of C.P. concentrated hydrochloric acid, 10 ml. of potassium dithio-oxalate solution, and 1.2 grams of sodium acetate, as described in the analytical procedure. Spectrophotometric measurements a t 508 mH, in I-cm. cells showed transmittances of 39 and 40%, which is in fairly close agreement with transmittances of 37 and 38% for test solutions containing 8 mg. of Sequestrene NA3 in 100 ml. of distilled water. A procedure based on calibrations employing urine dilutions
For many helpful suggestions the writer is indebted to H. ff. Zussman, mho instigated this work. Thanks are due to A. A. Dolnick for helpful criticism. LITERATURE CITED iklI
Bersworth Chemical Co., Framingham, Mass., Tech. Bull. 1, 17
(2)
“Lange’s Handbook of Chemistry.” 6th ed., Ohio, Handbook Publishers, 1946.
(1949). p. 1125,
Sandusky.
~ 3 Ibid., ) p. 1133. (4) Ibid., p. 1134, (5) Mellon, M. G., editor, “Analytical Absorption Spectroscopy,” p, 339, New York, John Wiley & Sone, 1950. ( 6 ) Plumb, Martell, and Bersworth, J. Phys. and Colloid Chem., 54, 5 (1950). RECEIVEDApril 12, 1951. Published with the permission of Alrose Chemical Co., Providence, R. I. Sequestrene is the registered trade-mark of Alrose Chemical Co. for ethylenediaminetetraaoetic acid and its derivatives.
Factors Affecting the Determination of ProteinBound Iodine in Serum JOHN J. MORAN’ S a n k Barbara Cottage Hospital Research Institute, Santa Barbara, Calif.
In order to ensure success with Chaney’s method for the determination of pmtein-bound iodine, a study was made of the factors involved. The chemistry of the reactions was studied, and the effect of variations in method and the kinetics of the ceric sulfate-arsenious acid reaction was determined. The resulting information makes possible successful use of the method, even by technicians with limited training in chemistr?..
F
OUR basically different methods for the determination of protein-bound iodine have been reported ( 2 , 6, 7 , l O ) . The method of Chaney ( 2 )has been successfully employed in this laboratory with some slight modifications, but in some cases it was apparently unworkable. Further investigation showed in these case8 an inadequate knowledge of the reactions involved or the factors influencing them. Accordingly, a study of the factors affecting Chaney’s method was undertaken, which ranged from the determination of impurities in the various reagents to the determination of constants of the ceric ammonium sulfate-arsenious acid reaction. The results are presented for the consideration of chemists, but should also lead to a greater degree of accuracy in the work performed by technicians of limited chemical training and ability. The data are applicable in varying degree to methods that employ precipitation of the bound iodine, acidic wet washing, distillation into a base, or the catalytic effect of iodide on ceric ion reduction by arsenious acid.
Sodium hydroxide, 1%. Phosphorous acid, 30y0 by weight. The prepared reagent 8u died bv J. T. Baker Chemical Co. has been found consistentK Ltisfaitory. Superoxol, 1 to 10 by volume. Arsenious acid, 0.064 N in 0.6 N sulfuric acid is prepared by dissolving 3.18 grams of reagent grade arsenious acid in 100 ml. of 1:4% sodium hydroxide in a 1-liter volumetric flask. The solution is diluted to about 500 ml. with water and 17.8 ml. of concentrated sulfuric acid are added. The solution is then made up to volume with water. Ceric ammonium sulfate, 0.012 AVin 1.6 N sulfuric acid is prepared by dissolving 7.16 grams of reagent grade ceric ammonium sulfate in approximately 900 ml. of 1.6 N sulfuric acid in a 1-liter volumetric flask and diluting the solution to volume with the acid. Slight precipitation will occur over a period of time, but this is not important if the clear supernatant liquid is employed. Standard iodide solutions. Standards containing 0.04, 0.08, and 0.12 microgram of iodide per ml. are stable for 2 to 4 months if kept in a cool dark place. PROCEDURE
REAGENTS
Redistilled water is distilled from an all-glass still containing a few pellets of sodium hydroxide. Unless otherwise indicated, all references to water imply redistilled water. Sulfuric acid, 0.67 N . Sodium tun state, 10%. Sulfuric acif, 70% by weight. Chromic acid, 60% by weight.
The following brief description indicates minor changes from the Chaney procedure in reagent strength or technique which the author has found advisable. Three milliliters of serum are added to a large centrifuge tube containing 3 ml. of 10% sodium tungstate solution and 20 ml. of water, 3 ml. of 0.67 N sulfuric acid are then added, and the mixture is well stirred with a rod. The precipitate is separated and is twice washed with 20-ml. aortions of water.. em~lovingcentrifugation. Twenty milliliters of 7070 sulfuric acid are added to the precipi_
Preaent address, Harold Wood, M.D. Pathology Laboratories, 1130 North Cen%?alAve., Phoenix, Aria. 1
I
-
V O L U M E 2 4 , NO. 2, F E B R U A R Y 1 9 5 2 tate. The well-stirred mixture is then poured into a digestion fiask containing a few glass beads and 2 ml. of 6OY0 chromic auid. The centrifuge tube is washed with 20 ml. more of 70y0sulfuric acid, which are added to the flask. The flask is placed on a hot heater. The mixture is brought to a boil and boiled for 3 minutes, after which the flask is removed from the hot plate and allowed to cool for 5 to 10 minutes. Then 20 ml. of water are added to the flask, which is replaced on the heater and allowed to boil until it is well filled with thin foglike fumes. The flask is removed from the hot plate and allowed to cool for 5 to 10 niinUtes, after which 10 ml. of water are added. The distillation apparatus is now washed with copious amounts oi water, care being taken that no air bubbles remain in the return tube after washing. The stopcock is closed and 1 ml. of 1% sodium hydroxide is introduced through the top opening; the return tube is avoided, so that the sodium hydroxide collects in and effectively blocks the trap. The flask is attached to the distillation apparatas and the side-necked dropping funnel with its stop cock closed is inserted into the flask. The entire apparatus is now lowered onto a hot heater, after which 10 ml. of 30% phosphorous acid are introduced into the dropping funnel. Violent bumping may occur during this initial phase of heatc ing, but tapping of the heater will help prevent it. After the mixture reaches a steady boil and before any distillate collects in the trap, the 10 ml. of 30y0phosphorous acid are quickly blown into the flask, immediately followed by 1 ml. of 1 to 10 Superoxol introduced in the same manner. Distillation is allowed to proceed for 10 minutes. At the end of this eriod the droppin funnel is removed, the entire apparatus liftegoff the heater, and the flask disconnected. The distillate is collected in a calibrated 15-ml. oentrifuge tube containing 0.25 ml. of the arsenious acid reagent and allowed to cool to room temperature. The volume is noted. The distillation apparatus is washed and is ready for the next distillation. One milliliter of 0.64 iV arsenious acid is added to each colorimeter tube; 4 ml. of water are added to the first tube which serves. as a blank for the ceric reaction, and 3 ml. of water plus 1 ml. of iodide standard are added to the next three tubes. The standards should contain, respectively, 0.04, 0.08, and 0.12 microgram of iodide. Four milliliters of distillate, in duplicates, are added to the remaining tubes. After thorough mixing the tubes are ready for the addition of the 0.012 N ceric ammodum sulfate solution and may be kept in this state for several hours. When ready, 1 ml. of the ceric solution is added to each tube a t exactly 30-second intervals and well mixed after each addition. The first tube ie read in the photometer (420 mp) exactly 20 minutes after the addition of the ceric ammonium sulfate and the subsequent tubes are read in order at 30-second intervals. CALCULATIONS
The optical densities of the standards are plotted against their concentrations on ordinary graph paper. The optical densities of the unknow-ns can then be translated into concentrations. Then, Concentration of unknown X volume of distillate X 100 = amount of distillate used X ml. of serum used iodine in micrograms per 100 ml. of serum
-.
From this value must be subtracted the amount determined for the reagent blank, obtained by running a blank through each step beginning with the digestion. The final value represents the amount of protein-bound iodine in micrograms per cent. TESTING OF REAGENTS
.4 convenient method of determining the purity of reagents takes advantage of the fact that the proportions of the reagents may be varied under proper conditions without influencing the accuracy of the over-all procedure. The purity of the reagents in the ceric sulfate-arsenious acid reaction is of prime importance. One and 0.5 ml. of arsenious acid reagent are added to colorimeter tubes containing, respectively, 4.0 and 4.5 ml. of water. 1 ml. of ceric ammonium sulfate solution is added to each and mixed well. An initial reading is immediately made in the photometer (420 mp), followed by readings at 10-minute intervals for 1 hour. There should be no appreciable decolorization in either tube, If decolorization occurs, interpretation is as follows: Equal decolorization in each tube indicates contaminated ceric ammonium sulfate, less decolorization in the second tube
379 indicates contaminated arsenious acid, and about 10% greater dccolorization in the second tube indicates contaminated water. Ksperience indicates that these reagents are not likely to be’ contitrninated. The precipitating reagents are t,ested by precipitating and washing (in the manner outlined in the procedure) 180 mg. of crystalline bovine albumin contained in 3 nil. of 0.9% sodium chloride and then carrying on the digest,ion, distillation, etc. The iodine value is compared with that obtained from 180 mg. of crystalline bovine albumin added directly to a digestion mixture. If contamination is demonstrated, addition of 3 ml. of each reagent to individual digestion flasks and subsequent determination of iodine will indicate the contaminated reagent. The testing must involve precipitation of proteins, as the washing of the precipitate efi‘ect,ively removes inorganic iodides not present in too large amount (Table I), whereas direct testing of the reagents may reveal iodide present in mild amount in the tungstate or in the SUIfuric acid, giving cause for needless worry. ~.
Table I.
~
-~ ~. ~
Effect of Different Precipitants on Recovery of Protein-Bound Iodine
(Deviations are standard deviations based on ten or more deterrninatlons in each category.) - ... lodide Mean Value Mean Value Retained Pooled Serum. after Adding in PrecipiPrecipitant Y ,% of and Washing tate, % Employed Iodide Ion 3 y of Iodide 7.4 i 0.3 1.4 6.0 i 0.2 Acetic acid 7.6 f 0.4 1.7 5 . 8 i 0.1 Zinc 2.0 8 . 1 =t0 . 5 Trichloroacetic acid 6.1 f 0.3 6.0 0.1 7.e 0.4 1.6 Tungstic acid
+
Sulfuric acid (70%) is easily tested by adding 20, 30, and 40 ml. of the acid to digestion flasks containing 2 ml. of 60% chromic acid and then continuing the procedure to final determination. In the author’s experience the chromic acid accounts for 80 to 95% of the blank and should be chosen with care. Technical grade flakes frequently show less contamination than reagent grades. The degree of contamination is ascertained by using 1 and 2 ml. of 60% chromic acid in the digestion blanks. Acceptable chromic acid contains not more than 3 micrograms % of iodine. The contamination is reduced to less than 0.5 microgram % by boiling 400 ml. of the 60% chromic acid to the spattering point in a 3-liter round-bottomed boiling flask containing glass beads. By successive addition of 300 ml. of water the boiling is repeated three times, and after the final boiling the acid is diluted to the original volume Kith water. Purity of the 1% sodium hydroxide is determined through the ceric sulfate-arsenious acid reaction. To colorimeter tubes containing 3.9 and 3.7 ml. of water, 0.1 and 0.3 ml. of the 1% sodium hydroxide are added, and 1ml. of arsenious acid solution followed by 1 ml. of ceric ammonium sulfate solution is added to each tube. Initial readings are made, followed by readings a t 10-minute intervals for 1 hour. Contamination of the Superoxol is determined by using 1ml. of a 1to 20 dilution and 1ml. of 1 to 10 dilution during distillation of reagent blanks. Experience indicates that this reagent is iodinefree. Purity of the 30% phosphorous acid is easily ascertained by using 8, 10, and 12 ml. during distillation of reagent blanks. FACTORS INFLUENCING THE DETERMINATION
Precipitants. Various means of precipitating the serum samples were tried. The acetic acid method detailed by Salter and MeKay ( 8 ) , the zinc precipitations described by Taurog and Chaikoff (11) and by Barker (I), trichloroacetic acid precipitation, and the method described in t,his report gave similar results (Table I). However, the use of trichloroacetic acid requires great care during the digestion to ensure removal of chlorides, which contribute to grossly erroneous results. The tungstic acid
ANALYTICAL CHEMISTRY
380 Table 11. Effect of Added Chloride on Iodine Determination (Deviations are standard deviations based on six or more determinations in each category.) NaCl Added t o Digestion Flask, Mg. 3 15 30
Mean Values of Iodide Ion, 5-minute 3-minute digestion digestion 7.1 =t0.7 12.8 f 0.8 10.4 f 1.1 1 3 . 0 f 2.1 12.2 f 1 . 3 14.2 f 1.6
y
% Complete digestion 5 . 4 f 0.2 5 . 7 f 0.1
5 . 3 zt 0.3
Table 111. Expulsion of Chlorides from Digestion Mixtures Containing No Iodine (Deviations are standard deviations based on ten or more determinations in each category.) Enhancing Effect of Chloride on 5.0 Micrograms % ti^^ of Boiling, of Iodide Ion Min. 1.0 mg. NaCl 5.0 mg. NaCl 20.0 mg. NaCl 0 6.4 f 0 . 3 9.6 zt 1.2 16.4 f 1.6 2 5.4 f 0.4 6 . 3 i 0.5 1 2 . 3 f 1.2 3 5.2 f 0.2 6 . 1 zt 0.1 8.4 f 0.4 Faint 801 fumes 5.0 zk 0 . 2 5.1 f 0.2 5.0 4- 0.1
precipitation was chosen because of its common use for other tests. It was found that two to three washings removed approximately 98% of 3 micrograms of iodide added to 3 ml. of serum before precipitation (Table I). Digestion. The digestion procedure is a frequent source of error for the analyst unfamiliar with it. The procedure entails digestion of the proteins with concomitant liberation of the bound iodine, which is then oxidized to iodate. The concurrent expulsion of chlorides must be complete or the iodine effect is enhanced. This enhancing effect was first reported by Sandell and Kolthoff (9) and examined in detail by Barker ( I ) , who employed it in the ceric reaction. The effect has been studied in this laboratory to determine fluctuations in apparent values caused by adding chlorides to the precipitated protein mass (Table 11). The expulsion of chlorides from digestion mixtures containing no iodine was then studied (Table 111). To the distillates were added equal amounts of iodide and the apparent values were noted against a standard containing no chloride. As a result, the digestion has of necessity two definite boiling periods. The first boiling serves to digest the greater percentage of the proteins and allows the iodine to be converted to iodate. Boiling is stopped as soon as the faintest fumes appear. Strenuous boiling is avoided, to prevent loss of iodine before it is converted to nonvolatile iodate. In the second period, however, strenuous boiling is encouraged and continued until the flask is well filled with fumes which are not overly dense. This serves effectively to expel chlorides while retaining iodates.
Table IV.
Loss of Protein-Bound Iodine with Prolonged Boiling of Digestion Mixture
(Deviations are standard deviations based on fifteen or more determinations in each category.) Duration of Boiling Past Faint SO! Fumes, Min. Iodide Ion, y % 1 5 . 0 f 0.3 3 4.1 f 0 . 5 5 3.6 f 0 . 3 10 1.4 i 0.2
The importance of stopping the boiling a t this point cannot be overemphasized. It is, in the author’s opinion, the most critical point of the entire determination. If the boiling is allowed to continue until very dense fumes fill the flask, loss of iodine will occur (Table IV). The loss is probably occasioned by a reduction of the iodate, for a t this point a partial reduction of the
chromic acid blank is evidenced by a slight greenish discoloration. It is unfortunate that no exact time can be assigned to the boiling periods, and each laboratory must determine for itself the time to be employed. Of prime importance for good duplicable results is a constant nonfluctuating source of heat. In this Iaboratory, Cenco Hotcone heaters were found far superior to gas or electric plate heaters. Distillation. Barker (1) and Taurog and Chaikoff (11) have discussed the distillation procedure. The former reported a recovery of 0 to 10% following Chaney’s method, while the latter reported an average recovery of 87y0 following the same method. Barker then substituted sodium sulfite for sodium hydroxide in the trap and subsequently had recoveries averaging 90%. Experiments were conducted in this laboratory to seek an explanation for the discrepancy between the two papers. Iodide (0.12 microgram) Fas added to 3-ml. aliquot8 of pooled sera which had a determined average value of 0.058 microgram per ml. The extent of recovery ITas then ascertained, employing varying concentrations of phosphorus acid with and without peroxide (Table V). The results ranged from a low (18 to 42%) recovery for the theoretical$amount of phosphorus acid used alone, to a high (88 to 94%) recovery for an increased amount of phosphorus acid with hydrogen peroxide. In each case the extent of recovery was roughly proportional to the time of distillation; this indicated that the iodate was not immediately reduced to hydriodic acid. The poor recoveries can be explained on the grounds that reduced iodate distilled as hypoiodite, iodine, and hydriodic acid, The first two react with Che sodium hydroxide in the trap to produce sodium iodate along with sodium iodide and sodium hypoiodite. Furthermore, the iodide and hypoiodite react with each other ( 4 ) ,liberating more iodine, which in turn reacts with the sodium hydroxide to produce more iodate. Under such conditions a substantial amount of iodate is present in the distillate. Table V.
Recovery of Added Iodide during Distillation
(Effect of varyin concentration of phosphorus acid, absence or presence of hydrogen peroxise, and varying duration of distillation. Added iodide varied from 0.03 to 0.30y. Range of percentage recovery is total range observed.) Recovery of Iodide Added to Precipitated Serum, % Duration 8 ml. 30% 10 ml. 30% Volume of of DistilHaPOs, HsPOs, Distillate, lation 8 ml. 30% l ml. 1-10 10 ml. 30% l ml. 1-10 M1. Min.’ HiPOs HZOI HsPOt HzOz 5-6 4 12-18 40-50 20-34 60-70 74-89 40-70 25-40 6-8 6 14-18 60-80 30-45 80-90 8-10 8 14-35 10-13 10 18-42 70-80 30-50 88-94
.
Experiments showed that iodate does not catalyze the ceric sulfate reaction as does iodide, strengthening the assumption that the distillate contained both iodide and iodate. The use of sodium sulfite in place of sodium hydroxide as proposed by Barker would eliminate this difficulty, but as the resultant sulfurous acid also reduced ceric sulfate, it had to be eliminated prior to the ceric sulfate reaction. Its removal was too cumbersome for routine use. The addition of arsenious acid to the sodium hydroxide resulted in a distillate which caused an immediate partial reduction of the ceric ammonium sulfate. The reduction proved to be independent of the amount of iodide present in the distillate and experiments along those lines were terminated. Thomas et al. (12) have published a modification employing arsenious acid and sodium hydroxide, which is apparently successful. In later experiments, conducted after publication of Thomas’ proposal, the same partial decolorization was obtained. Therefore the author feels that the original decision to focus attention upon the complete reduction of iodate prior to distillation was the only satisfactory ansn-er to the problem of obtaining high yields. In practice, a ratio of about 3 grams of phosphorus acid to
V O L U M E 2 4 , NO. 2, F E B R U A R Y 1 9 5 2 2 grams of chromic acid assures adequate reduction of the iodate in the digestion flask. It appears that 100% recovery is impossible, as almost half of the reduced iodate is distilled in the first few minutes and a very small amount is distilled as iodine, resulting in the formation of iodate. As the use of peroxide increases the recovery, in this instance it must act as a reducing agent, reducing liberated iodine to hydriodic acid in the digestion flask. 0 5(
6 4 . y
04C
03C 0.2c
38 1
Catalytic Reaction. The reduction of ceric ammonium sulfate by arsenious acid is complex, Two salts of ceric ammonium sulfate exist, one yellow and the other orange yellow (6). The ratio of one to the other is not fixed, but is dependent upon the mode of preparation. In addition, it appears that in solution a highly colored intermediary reduced form exists (6). This reduced form, cerosoceric hydrosulfate, does not occur in a known definite percentage nor is it readily related to the amounts of ceric and cerous sulfate present, although some definite relationship must exist. This is substantiated by the fact that optical densityconcentration curves for ceric ammonium sulfate a t different temperatures (Figure 1)form a family of curves and, given several of these curves, it is possible to plot a curve for an arbitrary temperature within 5 to 10% of the experimental curve for that temperature. During catalysis the state of the reagents is in flux and it has been impossible in this laboratory to determine the exact composition of the state a t a given time. The only statement that can be made a t present is that the reduction proceeds in an orderly although complex fashion to finality when a colorless solution of cerous sulfate exists.
0.1(
Table VII. Pseudo-First-Order Rate of Ceric Ammonium Sulfate-Arsenious Acid Reaction, and First-Order Effect of Iodide’Concentra tion 2 -0
40
60
a 0 ..
100
Figure 1. Relationship between Optical Density and Concentration of Ceric Ammonium Sulfate at Varying Temperatures
If it is postulated that the iodate forms complex acids with the chromic acid, it is possible to understand why the iodate is not quickly and completely reduced to hydriodic acid in the presence of a powerful reducing agent. It is equally evident that the iodate may then be distilled as hypoiodite, iodine, or hydriodic acid. Although this deduction was not made by Barker, data in his paper corroborate this point. The discrepancy between Barker’s recovery of 0 to 10% when employing sodium hydroxide and Taurog’s and Chaikoff’s recovery of 87% can then be explained, In Barker’s case the reduction of iodate was not complete and resulted in the distillation of hypoiodite which was converted by sodium hydroxide to iodate, whereas in Taurog’s and Chaikoff’s case the distillation product was hydriodic acid (with some iodine), resulting in the formation of iodide. Barker’s reagents differ from Chaney’s in that the former are upon a weight per volume basis and the latter’s are on a weight per weight basis. Thus Barker’s reagents are appreciably weaker than Chaney’s. A final factor influencing the recovery is the rate a t which the digestion mixture is boiled during distillation. I t would appear that a slow moderate rate would give best results, in so far as it permits complete reduction of the iodate. Such is not the case (Table VI).
Table VI.
Effect of Varying Rate of Distillation on Recovery of Added Iodide
(Added iodide varied from 0.30 to 0.30 y. Range of percentage recovery is total range observed) Recovery of R a t e of Distillation Added Iodide MI. Min. Ion, % 12 6 78-92 12 88-94 10 12 65-85 20
Summarizing, phosphorus acid should be employed in excess and should be quickly added after brisk boiling has set in, the use of hydrogen peroxide should not be disregarded, and the distillation should be briskly carried on for an adequate time.
0.04 y
%.
Times, Sec. 300 600 900 1200 1500 1680 AY. K
(Temp. 19.0’ C.) Concentration of Iodide - 0.08 y
ceric ion in soluK X tion 1085 0.58 72 0.55 64 0.50 54 0.53 47 0.50 42 0.52 (0.53 =k 0.016) X 10-3
n
ceric ion in K X solution 10-3 73 1.05 55 1.00 42 0.97 29 1.06 22 1.01 18 1.02 (1.02 i 0.015) X 10-3
0.12 y
%.
ceric Ipn in
K X tion 10-8 61 1.64 39 1.57 24 1.59 14 1.68 8 1.66 6 1.67 (1.64 zt 0.034) X 10-s 80111-
In view of the foregoing, it is understandable that dilutions of ceric ammonium sulfate do not follow Beer’s law (Figure 1). However, with such a curve, optical density can be translated into Der cent of ceric ammonium sulfate oresent. With this 2.303 where a = information and the equation K = tlog
a
a--2
initial concentration, a - x = concentration after time t , and K = velocity constant, it can be demonstrated that the arsenious acid-ceric sulfate reaction is pseudo-fist order (Table VII), if the arsenious acid is present in excess. From Figure 1 it is apparent that there is no special reason why the reaction must be run a t an elevated temperature if blanks and standards are included; therefore it is the practice in this laboratory to run the reaction a t room temperature, It is important, however, that the standards and unknowns be a t the same temperature, since the optical density of the solution is increased by an increase in temperature. This can be achieved by letting the tubes stand in racks under identical conditions for approximately 20 to 30 minutes prior to the addition of ceric ammonium sulfate. Direct sunlight will catalyze the reaction, even in the absence of iodide, and proper precautions must be taken to prevent this effect. It has not been found necessary to equilibrate the colorimeter tubes as suggested by Salter and McKay ( 8 ) , and minor fluctuations in ionic strength appear to have no effect. Because standards are used, it is not necessary to standardize the arsenious acid against the ceric ammonium sulfate. The reaction is most sensitive if the normality of the arsenious acid is approximately 5 times that of the ceric ammonium sulfate. Therefore the strength of the ceric ammonium sulfate solution may be changed to meet the requirements of other types of photometers if the
ANALYTICAL CHEMISTRY
382
iatio is maintained. Very poor and erratic results occur if the reagents are of equal normality. The best curve for routine use is obtained by using 0.04, 0.08, and 0.12 microgram of iodide (Figure 2). The slope of the curve will depend upon the reaction time and temperature. The average room temperature in this laboratory is 21' C. and the reaction time employed is 20 minutes. The curve is made b~ drawing a straight line between the points. Maximum deviution from the true curve is about 0.002 microgram. The curve, as given, is in the most accurate portion of the photometric readings, and therefore extension is not warranted. Furthermore, points based on 0.16, 0.20, and 0.24 microgram of iodide are so close a t 20 minutes that accuracy in this range is nil. On the other hand, readings taken say a t 10 minutes, when accuracy still exists in the high range, give very little accuracy in the range 0.01 to 0.08 microgram. This difficulty cannot be overcome by running the reaction a t a higher temperature. It is possiblr, of course, to take readings a t both time intervals, but higher stnndaids niust then beincluded. It is the practice in this laboratoiy to dilute samples that give very high results and make a second determination.
this line a first approximation for the relationship of concentration to optical density can be derived. The equation, y = 1.74 (z)0*67 (where y = optical density and z = concentration in yo), is only approximately correct and does not hold in either the high or low values of 2. On the other hand, the plotting of the reduction of the ceric ion (Figure 5 ) also presents a parabolic curve for the various concentrations of iodide. These latter curves are dependent upon the rate of the reduction, K , which is in turn dependent upon the concentration of iodide (Table VII) and
Figure 3. Log of Rate of Reaction K Plotted against Reciprocal of Absolute Temperature, Projected to Determine Rate of Reaction at 0" c.
0
IODIDE ION, MICROGRAMS
Another source of error is in the conatruction of a nomogram as detailed by Salter and hfcKay (8). It is claimed that surh plottings show the rate of reduction of the colored ion a t any given concentration of iodide to be a linear function. Work done in this laboratory does not verify that statement. Instead, the function is ct definite curve. It is true that the reduction of ceric ammonium sulfate ia pseudo-first order, but this holds only if the arsenious acid is in sufficient excess. The mathematical determination of the rate of reaction is dependent upon the actual concentration of ceric ion a t any given instant. This concentration can be obtained for a particular temperature by the construction of a curve as in Figure I, where known concentrations are plotted against their optical densities. Furthermore, the log of the rate of reaction K can be plotted against the reciprocal of the absolute temperature (Figure 3) and the resulting straight line establishes the firstorder nature of the reaction. The effect of iodide concentration within the limits 0.01 to 0.15 microgram is first order, but deviates from first order outside these limitations. It is important to remember that the concentration of the ceric ion does not obey Beer's law. From Figure 1 it can be seen that the curve of the plot is parabolic, and by taking the log of the concentration and plotting it against the log of the optical density a slightly curved line can be drawn (Figure 4). From
temperature. If nov, a composite graph showing the curve ( A ) for the relationship of ceric concentration against optical density and a standard curve ( R ) showing the reduction of ceric ammoniumsulfate ion under catalytic effect be drawn (Figure ti), the one is a very rough reciprocal of the other. It is this canceling out effect which gives a semblance of linearity as drawn in Salter's nomogram. Because the concentration-optica1 density curve is comparatively constant (Figure 1) whereas the concentration-catalysis curve is sensitively subject to temperature, it can be shown that a t room temperature the construction of
J
.5
I .2
c.4
-v
c n
0
0.6 LL
0
0.3
0.5 I .o I .5 2.0 Figure 4. Log of Optical Density Plotted against Log of Concentration
V O L U M E 2 4 , NO. 2, F E B R U A R Y 1 9 5 2
383 Kl and Kz are the rates at the absolute temperatures T1 a i d 'f-.xrid R is the gas constant in calories. A S , the entropy of activation, is obtained by the equation KO = 5.7 X lo1* X ,-&IRT X (3). The change in free energy, AF, is obtained from 18' = AH - T S S . T l i ~temprrature coefficient for a IOo intei~valis derived from
i l l i$-lii(,li
Table VIII. Constants of Ceric .Ammonium Sulfate.\rsenioue Acid Reaction at Varying Temperatures [odide Ion.
K
Temp.,
C. 16.8 19.4 30.0 37.2 16.8 19.4 30.0 37.2 16.8 19.4
0.04
0 08
0 12
AF, Cal.
X
10-3
O
e ,
3Iole-1 21,440 21,510 21,970 22,270
0.46 0.511
0.93 1 ,3 3
30.0 37.2
0.92 1.04 1.82 2 61
?i,ozo 21,110
1 40 1 61 2 88 4 o:
