V O L U M E 2 4 , NO. 2, F E B R U A R Y 1 9 5 2
373
Table 11. Determination of Copper i n Iron with Citrate Complexing Agent Iron Sample Grams'
Copper Added, hIg.
Copper Found, Mg.
Deviation, hlg.
tion is 0.04 microgram of copper. Using 1 microdrop of n-hexyl alcohol as extractant, the limit of identification is 0.03 micrograni of copper. The limit of concentration in the former case is I to 1,250,000 and in the latter case 1 to 1,660,000. Therefore neocuproine is found to be as sensitive as dithisone ( 3 ) . QUANTITATIVE DETERMINATION OF COPPER IN PRESENCE OF IRON
Spectrographic determination.
complex which was extractable under the conditions employed. The common anions chloride, sulfate, nitrate, perchlorate, tartrate, citrate, and acetate do not interfere. An orange-yellow precipitate occurs with large amounts of nitrate, perchlorate, and the halides (particularly iodide) upon the addition of neo-cuproine to an aqueous copper solution but causes no interference, as the colored complex is quantitatively extracted in all cases. Anions such as periodate, nitrite, thiocyanate, and ferricyanide, which either react with hydroxylamine or give a yellow colored solution, may be suitably eliminated to adapt conditions to the test, Pyrophosphate and phosphate ions do not interfere, although for excessive amounts two or three extractions may be required to collect all the copper. Neo-cuproine may thus be said to be completely specific. LIMIT OF QUALITATIVE IDENTIFICATION
Distribution coefficient measurements on the complex obtained using n-amyl alcohol, isoamyl alcohol, and n-hexyl alcohol show that n-hexyl alcohol is definitely the best extraction solvent. Employing visual examination of the aqueous droplet of colored solution in a spot plate without extraction, the limit of identifica-
Using citric acid as masking agent for ferric and ferrous ii nii a t pH 4 to 6, a twofold excess of hydroxylamine hydrochloriilo its reductant, a 100% excess of neo-cuproine as copper reactant, m i l 10 ml. of n-hexyl alcohol as extractant, results were obtaine(1 as shown in Table 11. Six grams of sodium citrate were added for each gram of iron present, but this amount is not critical. Fluoride, pyrophosphate, oxalate, malonate, and tartrate were studied as potential masking reagents for iron in the determination, but citrate proved t o be far superior within the pH range desired. PROPOSED EXTENSION IN APPLICATION OF NEO-CUPROINE FUNCTIONAL GROUP
It has been shown that substitution of methyl groups in thc 4,7positions of the 1,lO-phenanthroline molecule exerts only a sinall shift in the wave length of maximum absorption but a pronounced increase in the molecular extinction coefficient of the ferrous raniplex (6). Therefore it is proposed to investigate the 2,4,7,!)-t~tramethyl-1 ,lO-phenanthroline derivative as a possibly improved copper specific. LITERATURE CITED
(1) Breckenridge, Lewis, and Quick, Can. J . Research, B17, 258 (1939). (2) Case, F. H., J.Am. Chem. Soc., 70,3994 (1945). (3) Fisher, Mikrochemie, 8,319 (1930). (4) Hoste, Anal. Chim. Acta, 4, 23 (1950). ( 5 ) Irving, Andrew, and Risdon, Nature, 161, 805 (1948). (6) Smith and Brandt, ANAL.CHEY.,21, 1313 (1949). RECEIVED May 9, 1951
Colorimetric Determination of the Sodium Salts of Ethylenediaminetetraacetic Acid ALBERT DARBEY Alrose Chemical Co., Providence, R. I .
E
THYLENEDIAMINETETRAACETIC acid (Sequestrene) is readily determined as its alkali metal salts in the absence of alkaline earth metals and heavy metals by titration with standardized calcium acetate solution; sodium oxalate is used as an internal indicator. The end point is the appearance of permanent turbidity. In the presence of metals this method would in general determine essentially the free compound. The alkali metal salts of ethylenediaminetetraacetic acid are widely used as alkaline earth and heavy metal sequestrants. In many cases, therefore, it is desirable to have methods of determining the total amount of the compound in very low concentrations in the presenceof certain metulswhich may have been sequestered by it. Nickel may be employed to displace from their ethylenediaminetetraacetic acid complexes such metals as calcium, magnesium, and many other common metals, the nickel itself being then preferentially sequestered (6). In agravimetricmethodemploying this principle ( 1 ) a known amount of nickel is added, and excess, unsequestered nickel is precipitated as the hydroxide and sub-
sequently converted to the dimethylglyoxime salt €or gravimctric determination. The difference, representing sequestered nickel, may be calculated to ethylenediaminetetraacetic acid, ab the reaction is stoichiometric. This gravimetric method, however, may not be suEcic.iitlv accurate when very small amounts of ethylenediaminetetraawtic acid salts are to be determined. The method described here offers a colorimetric procedui P by means of which total ethylenediaminetetraacetic acid in very low concentrations may be quantitatively determined in the pre-ence of certain metals. Nickel is employed to displace from their Sequestrene chelates such metals as calcium and magnehrn. The unsequestered nickel is removed from the sequestered nickel by a modification of the method of precipitation with dimethylglyoxime ( 3 ) ; then, as the nickel chelate is highly dissociated in strongly acid solution, it becomes possible to liberate the sequestered nickel and determine it colorimetrically in acid .solution. The amount of nickel sequestered is related to the aniiiunt of ethylenediaminetetraacetic acid present. Potassium dithb-
ANALYTICAL CHEMISTRY
374 The research was iindertalcen to develop a more sensitive method for the determination of small amounts of total ethylenediaminetetraacetic acid salts in the presence of calcium, magnesium, iron, and certain other metals. Commonly employed methods are not sufficiently accurate for low concentrations. In the described method, which is sensitive to 0.2 to 0.5 mg. of the compound in 100 ml. of sample solution, the test solution reacts with a nickel salt. The nickel is selectively sequestered. Excess nickel is removed with dimethylglyoxime and the sequestered nickel is liberated a t low pH and
oxalate gives a bluish-red color with nickel in acid solution (4) and was found to be a suitable reagent for colorimetrically determining the nickel liberated from the complex. REAGENTS AND CHEMICALS
Sickel solutionl 13.3 grams of C.P. nickelous sulfate hexahrdrate per liter of distilled water. C.P. dimethylglyoxime, 1.5% in absolute ethyl alcohol. Potassium dithio-oxalate (Eastman), 0.25% aqueous solution, freshly prepared. C.P. aqueous ammonia, 28%. C.P. concentrated hydrochloric acid. C.P. calcium acetate monohydrate. C.P. ferric ammonium sulfate dodewhydrate. C . P . sodium acetate trihydrate. C.P. magnesium chloride hexahydrate. Commercially pure Sequestrene YA3 (trisodium salt, AIrose Chrinical Co.).
_ _ Table I. Transmittance of Two Sets of Test Solutions Test Solution Seu>irstreneNA3
jig.
8 4
+ 7 mg. of calcium +12 mg. of ferric ammonium sulfate +15 mg of magnesium chloride
1 4 4
4
Tlansmittance, %
Average Optioai Denalty
36 61 89 60
38
0 43
61 89 62
0 215 0.05 0 215
60 60
61 61
0 218 0 218
BASIS FOR T H E METHOD
Experimental test solutions were prepared by diluting 8, 4, 2, 1, 0.5, and 0.0 mg. of Sequestrene NA3 to 100 ml. in each case witli distilled water. Two other solutions were also prepared; one contained 8 mg. of Sequestrene NA3 and 10 mg. of calcium as calcium acetate in 100 ml. of distilled water, and the other contitined 8 mg. of Sequestrene NA3 and 15 mg. of ferric ammonium bulfate in 100 ml. of distilled water. To each solution were added 15 ml. of nickel solution and 6 ml. of C.P. aqueous ammonia (2876). To these solutions, in each case, were added 15 ml. of dimethylglyoxime solution to precipitate free nickel. After 2 to 3 minutes the dimethylgIyoxime nickel complex was removed by filtration, and to 50 ml. of each filtrate, 2.5 ml.of concentrated C . P . hydrochloric acid and 10 ml. of the potassium dithio-oxalate solution were added. A red to pink color resulted, depending on the amount of Sequestrene NA3 present. An 8-mg. test solution gave a red test color. A 0.5-mg. test solution showed a yellowish pink color. An 8-mg. test solution containing 10 mg. of calcium as calcium acetate or 15 mg. of feiric ammonium sulfate showed the same intensity of red color ap an 8-mg. test solution without these added metals. Testing of the filtrates (before addition of potassium dithiooxalate) for free nickel by addition of more diniethylglyosime showed no further precipitation of nickel. It is therefore apparent that the sequestered nickel is not precipitated by dimethylglyoxime, and that it can be subsequently lib-
determined colorimetrically as the red nickel dithio-oxalate. The intensity of the red color is related to the amount of sodium ethylenediaminetetraacetate. Because this sequestering agent is increasingly being used for w-ater softening, for clarifying commercial soap solutions, for biochemical applications and, in general, for inactivating heavy and alkaline earth metals, a method for its determination in low concentrations in the presence of heavy metals, alkaline earth metals, and some other substances becomes of some importance. The method can be adapted to a variety of problenis.
erated for colorimetric determination with potassium dit hiooxalate and thus the amount of ethylenediaminetetraacetic acid can be estimated. These experiments indicated further that ethylenedianiinetetraacetic acid may be quantitatively determined in the presence of alkaline earth metals, ferric iron, and nickel by measuring the intensity of the red color obtained in the test procedure This may be done by comparison with suitable color standards or by colorimetric measurement. The method h a q been adapted to both procedures. SENSITIVITY
The test color results indicate thpt the niethod will detect 0.5 nig. (or less) of ethylenediaminetetraacetic acid in 100 nil. of test solution. SPECTROPHOTOMETRIC .MEASUREMENTS
Instrument. A Beckman Model B spectrophotometer employing 1-cm. matched methacrylate plastic cells was the test instrument. Hydrion short range pH papers were used for pH determinations. Spectral Transmittance of Test Color. The red color obtained under the test conditions with nickel and Dotassium dithiooxalate shows maximum absorption a t 508 to 5f0 m p (Figure 1). Test Solutions for Spectrophotometric Data. Solut,ions were
W 0
z 4
I-
C r to
z U
a
I-
C,w 0
a W
a
450
500
WAVE
560
LENGTH
600
IN
650
H/J
Figure 1. Absorption Spectrum of Nickel Dithio-oxalate Solution 50 ml. of distilled water, 0.0026 g r a m of nickelous sulfate hexahydrate, 0.7 ml. of concentrated hydrochloric acid, 10 d. of potassium dithio-oxalate solution, 1.0 gram of sodium acetato pH 4 to 4.5 1-cni. cell
V O L U M E 24, NO. 2, F E B R U A R Y 1 9 5 2
375
made up to 100 ml. with distilled water containing 8, 4, 1, and 0.0 mg. of Sequestrene NA3, respectively. A 100-ml. solution containing 4 mg. of Sequestrene N.43 and 7 mg. of calcium as calcium acetate, a 100-ml. solution containing 4 mg. of Sequestrenc NA3 and 12 mg. of ferric ammonium sulfate, and a 100-ml. solution containing 4 mg. of Sequestrene NA3 and 20 mg. of magnesium chloride were prepared. These test solutions were treated as described below. The blank solution without Seqestrene N.43 was used as a reference set a t 1 0 0 ~ transmittance o (0optical density). Transmittance measurements were made a t 508 mp. Tuble 1 shows the transmittance of these test solutions arid that calcium, msgwsiuni, or ferric ions do not interfere with the determination. Figure 2 is a plot of transmittances and Figure 3 is a plot of the optical density data shown in Table I.
transmittance was shown in each case-Le., adding nickel to an almost neutral solution containing ethylenediaminetetraacetic acid gave the same transmittance as that obtained by adding nickel to an ammoniacal solution. However, in the presence of calcium the displacement of the calcium and selective sequestering of the nickel occurred more completely and rapidly a t initial test solution pH of 6 to 6.5 than in ammoniacal solution (Table 11). Therefore, to ensure completeness of formation of nickel Sequestrene the nickel should be added to the teet solution and held for 10 minutes before the addition of ammonia. Test solutions are adjusted to p H 6 to 6.5 before and not after addition of nickel 9olution.
INFLUENCE O F pH
Sequestering of Nickel. Experiments were run in which the nickel solution was added to the test solution before the addition of ammonia (as in the above experiments) and compared with test solutions to which ammonia was added before addition of the nickcl solution. In the absence of alkaline earth and heavy metals the same
Yz a + I-
zcn
CONC. SEQUESTRENE NA3
z a
I N 100 ML. SAMPLE SOLUTION Figure 3. Typical Optical Density Calibration Curve for Nickel Dithio-oxalate
U I-
t-
2
W
0
a W a
2ot
A s measure of Sequestrene NA3 concentration in 100 ml. of aample solution at 508 m
2
4
6
SEQUESTRENE NA3-
IN 100
HL. SAMPLE
8
HO.
SOLUTION Figure 2. Typical Transmittance Calibration Curve for Nickel Dithio-oxalate As measure of Sequestrenc NA 3 concentration i n 100 ml. of sample solution at 508 m p in 1-cm. cells
Table 11. Effect of pH on Displacement of Calcium from Sequestrene by Nickel Transmittance, 31g. 4
4 -7
7
of calcium
4
T
7 mg. of calcium
4
trig.
p
in 1-cm. cells
Table 111. Influence of Sodium Acetate in Delaying Development of Turbidity as Shown by Transmittances
CONC.
Test Solution Fequestrene N.43
- HG'
Procedure Sickel solution added t o test solution a t p H 6.5 and allowed t o react 10 minutes before addition of ammonia Nickel solution added t o ammoniacal solution and allowed t o react 10 minutes Kickel solution added t o test solution a t p H 6.5 and a1lowed t o react 10 minutes before addition of ammonia Xickel solution added to aminoniacal solution ( p H 11) and allowed to react 10 minutes
70 61
til
60
80
(Test solution, 8 my. of Sequestrene "43) ' .___ Transmittance, % After AfteFTest Solution Initial 10 min. 30 miri. N-itliout sodium acetate, p€J 1 37 26.5 21 With sodium acetate, p H 3.0-1 28-39 40 39
Liberation of Sequestered Nickel. A pH of 1 to 1.5 is necessary to liberate the sequestered nickel effectively, and the full amount (2.5 nil.) of hydrochloric acid must be used. .4n inwfficient amount of hydrochloric acid will delay the full develoiiment of the red color. illaximuni color development results when the hydrochloric acid is allowed to react with the sequestered nickel for 5 minutes before the addition of potassium dithiooxalate. Stability of Test Solutions. The color shows adequate stability to light, although turbidit,y gradually develops in the colored test solutions on standing. Development of pronounced turbidity would cause errors in colorimetric or spectrophotomet.ric measurements and in general would necessitate tht. preparation of a fresh test solution. The turbidity is probably due to some decomposition of the free potassium dithio-oxalate in strongly acid solution. Weaker acids than hydrochloric acid, such as acetic acid, were tried, but they do not effectively liberate the sequestered nickel.
376
ANALYTICAL CHEMISTRY Transmittances of Test Solutions Containing Sodium Acetate at pH 3.5 to 4
Table IV. Test Solutions, Mg. 8 4
1
Initial 38 62 87
Transmittance, % After 10 min. After 30 min. 40 39 62 58 85 83
The development of turbidity is delayed considerably if sodium acetate is added to raise the p H to 3.5 to 4 after the nickel has been liberated and the color allowed to develop a t pH 1. This is illustrated in Table 111. Table IV gives transmittances initially and after standing of 8, 4,and 1mg. of Sequestrene K'A3 test solutions adjusted to pH 3.5 to 4 with sodium acetate. It is apparent that there is ample time in which to determine transmittancy before the development of turbidity; however, the determinations should be made as soon as the test solutions are ready for measurement. In some cases it will be advantageous to prepare a calibration curve from known test solutions which have been clarified by suction filtration through asbestos after the turbidity has been allowed to develop for 30 minutes. Spectrophotometric measurements are made immediately on the filtered test solutions. The unknown in this case is likenise filtered before measurements are made. Filter paper cannot be employed, because the color is adsorbed to a great extent by the paper. PROCEDURE
To a sam le adjusted to pH 6.5 (using Hydrion short-range pH papers), anfdiluted t o 100 ml. with distilled water at 23' to 24OC., add 15.0 ml. of nickel solution. The amount of Sequestrene present must be within the calibration range (see Figure 2). Allow the reaction to proceed for 10 minutes, then add 5 ml. of C.P. aqueous ammonia (2870). After 5 minutes add with stirring 15.5 ml. of dimethylglyoxime solution. After 5 minutes filter through No. 40 Whatman filter paper. To 50 ml. of the filtrate add 2.5 ml. of C.P. concentrated hydrochloric acid. The resulting pH should be 1. After 5 minutes add 10 ml. of freshly prepared potassium dithio-oxalate solution. After 3 to 4 minutes introduce 1.2 to 1.4 grams of solid sodium acetate crystals with stirring to give a pH of 3.5 to 4. Make the spectrophotometric measurements immediately a t 508 mp in I-em. cells using a blank, as reference, set a t 100% T (0 optical density). The blank consists of 100 ml. of distilled water run through the same procedure as the sample. The graphs in Figures 2 and 3, representing typical calibration curves, are based on 100 ml. of sample solution with a blank as reference and may be used to obtain the concentration of Sequestrene in the sample. Commercially pure Sequestrene KA3 (trisodium salt of ethylenediaminetetraacetic acid with 1 mole of water, molecular weight 376) has been used in this work. The procedure is also applicable to the determination of the mono-, di-, and tetrasodium salts. The results in terms of anhydrous Sequestrene NA3 may be converted in accordance with Table V.
Table V.
Conversion Factors (for Anhydrous Compounds)
Mg. of Sequestrene NA3 X 0.8156 = mg. of ethylenediaminetetraacetic acid hlg. of Sequestrene NA3 X 0.8770 = mg. of monosodium ethylenediaminetetraacetate Mg. of Sequestrene N.13 X 0.9386 = mg. of disodium ethylendiaminetetraacetate Mg. of Sequestrene N.43 X 1.0614 = mg. of tetrasodium ethylenediaminetetraacetate
Calibration curves which would pertain directly to the particular salt originally present could be prepared from knowns containing any one of the sodium salts. It is recommended that the user prepare his own calibration curves from a series of knowns as previously described. In this way differences in equipment, technique, etc., are largely com-
pensated for. For the most accurate results, in special cases, it might be advisable to run two or more known test solutions along with the sample and the blank. DISCUSSION
Because this method is an empirical one, exact directions must be followed or exact proportional aliquots of all volumes used. If it is desired to have the major portion of a calibration more closely approach a transmittancy range of 10 to 50%, a range which in a general way is considered the most reliable spectrophotometric range (6),graphs may be prepared from measurements in cells of greater length than 1 em?, to obtain higher absorbance.
Table VI.
Reproducibility as Shown by Transmittances of Five Sets of Test Solutions
Test Solutions, Mg. 8
Transmittance, % Set 1 Set 2 Set 3 Set 4 36 37 38 38 4 61 60 60 61 1 89 89 88 88 Blank set a t 100% transmittance.
Set 5 38 62 87
Reproducibility. Satisfactory reproducibility of results is achieved by carefully following the analytical procedure outlined. This is illustrated in Table VI, which gives the transmittance of five sets of test solutions. INTERFERING SUBSTANCES
Phosphates. The presence of phosphates gives high results in terms of ethylenediaminetetraacetic acid, as these compounds sequester some of the nickel, which on subsequent liberation would be interpreted colorimetrically as corresponding to Sequeetrene NA3. Interference by small amounts of orthophosphates can be avoided by precipitation as calcium phosphate according to the procedure below. REAGENTS.Calcium acetate, reagent grade, 44 grams per liter of distilled water. Other reagents same as given above. To 98.5 ml. of clear sample adjusted to pH 6.5 PROCEDURE. add 15.0 ml. of nickel solution. Allow to stand for 10 minutes, add 1.5 ml. of calcium acetate solution, and then add 5 ml. of C . P . ammonia (28%). After 5 minutes filter from the recipitated calcium phosphate. To 60 ml. (one half of originaP) of filtrate add 7.5 m]. of dimethylglyoxime solution and after 5 minutes filter through So. 40 Whatman filter paper. To 50 ml. of filtrate add 2.5 ml. of concentrated hydrochloric acid (pH should be 1); after 5 minutes add 10 ml. of freshly prepared potassium dithiooxalate solution, and after 3 to 4 minutes introduce 1.2 to 1.4 grams of solid sodium acetate crystals with stirring to dissolve (pH 3.5 to 4). Make spectrophotometric measurements immediately a t 508 mp in 1-em. cells, using a blank as reference. In order to use the typical calibration curves DISCUSSION. (Figures 2 and 3), it is necessary to correct for the volume of 'added calcium acetate. As Figures 2 and 3 are based on a volume (sample-water) of 100 ml., the volume of added calcium acetate solution must be part of the 100*ml. In this particular case .the actual sample (sample-water) volume would be 98.5 ml. (plus 1.5 ml. of calcium acetate solution, 100 ml.). This must be taken into consideration when making calculations. A test solution containing 4 mg. of Sequestrene NA3 and 10 mg. of anhydrous disodium phosphate in 98.5 ml. of distilled water treated as above showed 62.5% transmittance. A test solution of 4 mg. of Sequestrene NA3 in 100 ml. of distilled water without disodium phosphate showed 61-5% transmittance. Citrates. Citrate if present as ammonium citrate has a slight sequestering effect on the nickel and would tend to give slightly higher results. However, if the citrate is present as sodium citrate, there is no sequestering of nickel under the test conditions. with 20 mp. of sodium citrate in 100 ml. of test solution. I
V O L U M E 2 4 , NO. 2, F E B R U A R Y 1 9 5 2 In the presence of up to 20 mg. of citric acid per 100 ml. of test solution, the citric acid is first neutralized to p H 6.5 with 0.5 N sodium hydroxide before addition of nickel solution. Also, as in the procedure for phosphates, the volume of 0.5 N sodium hydroxide added in this case must be allowed for in making up the 100 ml. of test solution and calculating the weight of the sample. A test solution containing 4 mg. of Sequestrene NA3, 10 mg. of sodium citrate, and 4 mg. of calcium as calcium acetate in 100 ml. of distilled water showed 62% transmittance in the test. A test solution containing 4 mg. of Sequestrene NA3 without calcium and sodium citrate showed 61% transmittance using a blank as reference. Copper. Copper seriously interferes with the method, because it f o r m brownish complexes with the reagents employed. The complex partially precipitates. For this reason, and because the copper niight displace some of the nickel in the test method, the setting up of a two-component color system (copper and nickel) would give very uncertain results. However, the copper can be removed and the test then carried out. 5,7-Dibromo-8hydroxyquinoline (2) was found to be ideally suited as a precipitant for copper in the test procedure. It precipitates copper in acid solution and its spectral absorption under the test conditions is nil a t 508 mp.
REAGENTS.5,7-Dibromo-&hydroxyquinoline (Eastman), 0.5 gram in 40 ml. of C.P. hydrochloric acid and 60 ml. of distilled water. Other reagents same as previously given. PROCEDURE. To 88.3 ml. of clear, neutral, or faintly acid (hydrochloric acid) sample add 11.2 ml. of 5,7-dibromo-&hy Iroxyquinoline solution. The precipitate forms slowly. Allow to stand 15 minutes before filtering. If precipitation of copper does not start within 5 minutes, add a small amount (0.2 gram) of sodium acetate to lower the hydrochloric acid content slightly. Filter, and to 80 ml. of the filtrate add C.P. ammonia (28%) to neutralize. About 3 to 3.5 ml. of ammonia will be required to obtain a pH of 6.5. Add 12 ml. of nickel solution and allow to stand 10 minutes. Add C . P . ammonia (28%)to give a total of 5 to 6 ml. of ammonia (about 2.5 ml. in addition to the approximately 3.5 ml. of ammonia required for neutralization) and bring the pH to 11. Add 12 ml. of dimethylglyoxime solution with stirring. After 5 minutes filter through No. 40 Whatman filter paper and to 50 ml. of filtrate add 2.5 ml. of C.P. concentrated hydrochloric acid (pH now 1). After 5 minutes add 10 ml. of potassium dithio-oxalate solution with stirring. Allow the color to develop for 2 to 3 minutes and then introduce 1.5 to 2 grams of solid sodium acetate to give a pH of 3.5 to 4 and determine transmittance a t 508 mp using I-em. cells. At the stage where the test solution is made DISCUSSION. alkaline with ammonia, the remaining excess of 5,7-dibromo&hydroxyquinoline is largely precipitated. This does not interfere with the precipitation of the nickel by dimethylglyoxime, both being filtered off together. Any 5,7-dibromo-8-hydroxyquinoline remaining in solution after addition of the ammonia will not influence the measurements because it does not absorb a t 508 mg. In the analytical procedure given, the volumes of reagents, sample, etc., have been so adjusted that the results of transmittance measurements may be compared directly with the typical calibration curves in Figures 2 and 3. The amount of Sequestrene NA3 found will pertain to 88.3 ml. of sample. A test solution containing 4 mg. of Sequestrene KA3, 10 mg. of cupric sulfate crystals, and 2 mg. of calcium in 88.3 ml. of distilled water and treated as above showed 62% transmittance, using as a reference a blank made up as above containing 10 mg. of cupric sulfate but without Sequestrene NA3 and run through the procedure in the same way as the test solution. The 62% transmittance is in agreement with 61% transmittance for a 4mg. Sequestrene KA3 test solution, obtained in the procedure in the absence of copper. COLOR STANDARDS FOR USE WITH NESSLER CYLINDERS
Suitable color standards may be employed in lieu of spectrophotometric equipment. The colors obtained in the test solu-
377 tions show some fading after several hours and therefore are not suitable as permanent or semipermanent standards. Color standards having reasonable stability were prepared with certain dyestuffs to match the colors of the following test solutions. Solutions containing 8, 4, 1, and 0.0 mg. of Sequestrene NA3 were diluted to 100 ml. and treated in accordance with the analytical procedure. In Table VI1 are given the compositions of color standards which match the test solutions when equal volumes are compared in matched Nessler cylinders.
Table VII. Composition of Color Standards Dyestuff Solutions. A. 0.050gram of Solantine Pink 4BL (Kational Aniline Division, Allied Chemical and Dye Corp.) (C.I. 353) per liter of,distilled, water. B. 0.0126 gram of Calco D.C. Brown No. 1 per liter of distilled water Composition of Color Standard, M1. Original Test Solution, Distilled hlg. A B water 45 2 53 8 18 2 80 4 6.5 3.5 90 1 0.3 4 96 0.0
The standards given in Table VI1 are illustrative. By following the same Ijrocedure, any number of intermediate standards may be prepared to cover any desired range. The standards are fairly close matches to the test colors. It is, however, very important that each user should check the standards against knowns. Corrections for any variation in dyestuffs or in individual technique, color discrimination, etc., may then be made by changes in the proportions of solutions A and B and water. The standards should be kept in closed glass bottles or Nessler cylinders in the dark when not in use. After 2 months the standards are somewhat paler and slightly yellower; therefore their color should be checked after 1 month and a new set prepared if necessary. The color standards must not be used in connection v-ith the spectrophotometer, as the coloring substances in the test solutions and in the color standards are not the same, and the spectral transmittance will not be the same. APPLICATIONS
The method is suitable for determining total ethylenediaminetetraacetic acid salts in industrial waters and solutions containing ethylenediaminetetraacetic acid with due regard given to p H and the possible presence of interfering substances or other nickelcomplexing compounds. In Alkali Metal Soaps. The following experiments indicate that the method is of value in determining Sequestrene NA3 in soaps. To 99.5 ml. of aqueous solution containing 4 mg. of Sequestrene S A 3 and 0.050 gram of Lux soap flakes was added 0.5 ml. Of.4.4% aqueous C.P. calcium acetate solution. The soap was precipitated as the calcium salt and was filtered off after 5 minutes. To 50 ml. of filtrate were added 7.5 ml. of nickel solution. After standing 10 minutes 2.5 ml. of c P. ammonia (28%) and 7.5 ml. dimethylglyoxime solution were added, and the samples were filtered after standing 5 minutes. To 50 ml. of filtrate were added hydrochloric acid, potassium dithio-oxalate, and sodium acetate as described in the analytical procedure. Transmittance measurements were made a t 508 m,u in 1-em. cells, using as a reference a soap solution made up without Sequestrene NA3, but carried through the procedure as above with the blank set a t 100% transmittance. The transmittance of the test solution which originally contained soap was 64%, while the transmittance of a similar solution which did not contain soap was 61.5%. The method can be adapted for soap plant control. For such applications preferably soap solutions containing known amounts of Sequestrene NA3 should be used in setting up calibration curves. In Urine. In connection with certain types of biological research employing ethylenediaminetetraacetic acid, a method for
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,
G., editor, “Analytical Absorption Spectroscopy,” New York, John Wiley & Sone, 1950. ( 6 ) Plumb, Martell, and Bersworth, J. Phys. and Colloid Chem., 54, (5) Mellon, M. p, 339,
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
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