and there would be a resultant loss of noradrenolutine fluorescence. Determination in Mixtures. T h e results of t h e determination of both the noradrenalin and adrenalin in a series of six aqueous solutions containing known amounts of these amines are presented in Table 11. For 0.03 to 1 y of adrenalin and 0.05 to 1 y of noradrenalin the recovery of the catechol amines is within lOyo of
the amount added. The lower limits of the method, for 20% accuracy, are Wproximately 0.01 y of adrenalin and 0.02 y of noradrenalin. ACKNOWLEDGMENT
The author would like to express his appreciation t o Hans Jeiisen and Ulrich Westphal for their interest in his work.
LITERATURE CITED
(1) Euler, U. S. v., Floding, I., Acta Physiol. Scand. 33, Suppl. 118, 45 (1955). (2) Lund, A., Acta Pharmacol. Tozicol. 6 , 137 (1950). (3) Mylon, E., Roston, S., Am. J . Physiol. 172, 612 (1953). (4) 11-eil-hlalherbe, H., Bone, -4. D., Bzochem. J . 51, 311 (1952).
RECEIVEDfor review, ~~l~ 31, 1957. Accepted RIarch 6, 1958.
Complexometric Titration of Calcium in the Presence of Magnesium ALEXANDER D. KENNY and VICTOR H. COHN Department o f Pharmacology, Harvard Medical School, and Biological Research laboratories, Harvard School o f Dental Medicine, Boston 7 5, Mass.
b By modifying the (ethylenedinitril0)tetraacetate complexometric titration method of Munson and his associates for the determination of calcium (20to 50-7aliquots), it has been possible to raise the limit of magnesium interference, arbitrarily taken as greater than 24/,, from a magnesium to calcium weight ratio of 3 to above 16. The modification entailed ensuring that the p H of the titration solution was 12.4 to 12.5. At lower pH values calcium was overestimated, and a t higher pH values it was underestimated.
A
and precise (ethylenedinitri1o)tetraacetate (EDTA) coniplexometric titration method for determining calcium has been developed ( 5 ) . Kenny and Toverud ( 4 ) indicated the noninterference of phosphate in a similar method in the presence of phosphorus to calcium ratios as high as 8. The degree of magnesium interference was studied so that the possibilities of the application of the method to the analysis of calcium. particularly in biological tissues, could be more clearly defined. K i t h the usual procedure, there was interference a t ratios of magnesium to calcium as lovi as 3; with the modified method, no important interference occurred with ratios as high as 16. RAPID
REAGENTS AND EQUIPMENT
Coleman junior spectropliotometer, using Evelyn colorimeter tubes (No. 4635, Rubicon Co., Philadelphia, Pa.) in a Coleman cuvette adapter (No. 6-102, Coleman Co., Inc., Los Angeles, Calif.). The adapter was turned on a lathe to enlarge the internal diameter for use with the Evelyn tubes. 1366
ANALYTICAL CHEMISTRY
A Beckman Rlodel G pH meter was used for p H determinations with the Beckman Type E glass electrode. EDTA solution, 0.18 gram of disodium dihydrogen (ethylenedinitril0)tetrancetate p e j liter of distilled water. Standard calcium solution. 10 nig. per 100 ml. Anhydrous calciuni carbonate, standard luminescent grade (llallinckrodt Chemical Korks, St. Louis, No.), was dried for 4 hours a t 130" C., and 0.2497 gram of t h r dried salt was dissolved in 25 ml. of water containing 2 ml. of concentrated hydrochloric acid and diluted to 1 liter with water. This was assumed to be a primary standard ( 1 ) . Stock magnesium solution, 100 mg. per 100 ml. Reagent grade magnesium sulfate heptahydrate m s used. \Torking magnesium solution, 50 and 5 mg. per 100 nil. Ammonium purpurate solution. The ammonium purpurate, 50 mg. (Hagan Corp., Pittsburgh, Pa.), was shaken n-ith 25 ml. of water sild filtered into a broivn bottle. Fresh solution was prepared after 2 days. 2-Octanol, 20% in 9.5% ethyl alcohol. All water used ]vas double-distilled. PROCEDURE
The E D T A complexometric titration method developed by hIunson and his associates ( 5 ) n a s used with a slight modification. The calcium solution to be analyzed vas unbuffered and a t a neutral pH to avoid the necessity of finding the precise amount of sodium hydroxide needed for a final pH of 12.4 to 12.5. The solution for titration was prepared in an Evelyn colorimeter tube by mixing a calcium aliquot of 20 to 50 y, varying amounts of working niagnesiuni solutions, water to n total volume of 9.0 nil., 2.0 nil. of 0 . 2 5 sodium hydroxide, 1 drop of 2070 2octanol, and 0.3 nil. of ainnioniuni purpurate solution. The final p H was
12.4 to 12.5. The colorimeter tube was immediately placed in the special cuvette adapter in the spectrophotonieter. A bubbling device and a buret with extended tip were lowered into the colorimeter tube. The solution was titrated in the spectrophotometer a t 520 nib. Nitrogen or air was bubbled in to mix the contents during each addition of 0.2 ml. of the EDTA solution. Readings of transmittance were taken after each addition. By plotting the points on seiiiilogarithmic graph paper, the end point n a s determined by the point of intersection of the two straight lines. There Ivere three or four replicates a t each ratio of magneqiuni to calcium. All values of calcium found were expressed as a percentage of the theoretical calcium contents follon ed by the standard deviation. The values which received suhsequcnt statistical treatment n-ere determined on random samples, the analyst being unanare of the code. RESULTS
The original procedure (6) rvas follo\ved in which the pH was not rigidIy controlled; 4 drops of 9 N sodium hydroxide n'ere addfd to the calciuni aliquot in a total volume of 10 nil. As the ratio of the neight of magnesium to calcium was increased from 0 to 10, the degree of interference Ivas greater. From the data of an experiment in which the aliquot of calcium \vas 50 y. the regression line, represented by the equation I' = 99.99 - 0.721X, was calculated. I t is plotted in Figure 1, experiment 1 together with the observed points. Extrapolation of the regression line, represented by the dotted line, is justifiable; in other experiments conducted under similar conditions. a magnesium to calcium
ratio of 20 for the gooled xiieaii of r i i w determinations rcsulted in 87.6 L 2.9% of the theoretical value. Andysis of variance sho\ved this regression to be significant' and t'he deviations from linearity t o be insignificant. If 98.070 is arbitrarily chosen as the point a t which the int'erference becomes iniport'ant, the regression line (Figure 1, experiment 1) shon-s that interference began at a magnesium to calcium ratio of 3. This n-as unsatisfactory because of the possibility of encountering such ratios in the analysis of soft tissues such as muscle. Various attempts to raise tlir limit of interference by modifying the original method were made. Increase of Purpurate to Calcium Ratio. Incicasing thf ammonium 1)urpuratc concentration tn-ofold by taking a 0.6- instead of a 0.3-nil. :iliquot o f dye solution resulted in less interference a t a magnesium t o calc*iuniratio of 10. r s i n g a 50-y aliquot of calcium and t'itrntiiig a t 505 nip(, thc mean values of quadruplicate deter0.470 of the iiiiriatioiis w r e 92.3 tlicoretical value for the 0.3-nil. aliquot of dye and 97.0 + 0.9% for the 0.6-nil. :iliquot' of dye in the presence of a iiiagriesiuni to calcium ratio of 10. Such n modification is liniited by the production of a colored mixture \\.hose light absorption is too great. It \vas :~b:tndonctl in favor of decrrasing t,he sliquot of calcium from 50 to 20 and 10 y. I-se of a 20-y aliquot of calcium resulted in less interference. The regression line (1- = 99.98 - 0.2939) calculatcd from the data of an experinient in n-hich the aliquot of calcium was 20 y is plotted in Figure 1, experinient 2 , toget'her n.ith the observed points. An analysis of variance showed the regression to be significant and the deviations from regressions to be insignificant. The dotted line represents the extrapolation of t'he regression line. I n other experiments conducted under similar conditions, a magnesium to calcium ratio of 20 resulted in a value of 95.1 =t 2.57,, the pooled mean of six determinations. K i t h a 20-7 aliquot of calcium, interference did not begin until the presence of a niagnesiuni t o calcium ratio of 7 , a considerable imi)rovement over the 50-7 aliquot. The fact' that the wave lengths used in experiments 1 and 2 were 510 and 505 m p , respectively, is not iinportant, st least with the spectrophotometer used. I n a separate experiment in which both 50- and 20-7 aliquots of calcium were each determined a t ii-ave lengths of 505 and 510 nip in the presence of magnesium, no significant cffect on the degree of interference attribut'alde to the difference in wave length n x s notcd. d 10-7 aliquot of calcium did not afford additioiial iniprovement and some precision iyas lost. I n a series of t'hree experiments using a
*
.a2
r
98
----- -
96 94
92
- EXPT.2 - -.
1 EXPT. I
'.'.
MAGNESIUM / C A L C I U M
RATIO
Figure 1. Regression lines for interference of magnesium in EDTA-calcium method
0 Experiment 1;
5 0 y calcium, p H
X = 5 1 0 mp, std. dev. 3~ 1.3% A Experiment 2; 2 0 y calcium, p H
>
12,
> 12, X = 505 mp, std. dev. f 1.2% Experiments 4 and 5; 50 y calcium, p H 12.4 t o 12.5, X = 5 1 0 mp, std. dev. k 1.7%
Table 1.
Effect of pH on Magnesium interference in EDTA-Calcium Method 11 8 12 5 12 4 12.8 PH Experiment 3 4 3 G
lIg./Ca Keielit
It;lt%,
Calcd.
Calcium Found," 5%
0 0.4 1
100.0 100.0 100.0 100.0 103.0 101.2 101.2 99.9 105 1 101.3 100.4 100.9
3
10
20
107.6 103.1
08.2 101.2 100.2 98.6 '38.8 '36.9 I06 fi 9 7 . 7 '37.2 0 0 . 1
' ,\liquets of 50 y of calcium were used. The titrations were conducted at a xave length of 510 n i p . Each value is the mean of three replicates.
10 y aliquot of calciuiii, a iiiagnesiuni to calcium ratio of 10 gave a pooled mean for 12 replicates of 97.7 k 2.07,, the higher standard deviation iiidicating some loss of precision. Effect of pH. A study of t h e effect of p H on t h e e-itent of magnesium interference yielded t h e most fruitful results. Instead of using t h e method developed b y Munson and his associates (S), nhich is sufficient for the determination of calcium in the presence of a magnesium to calcium ratio of 1 or less, a procedure n as followed which had better pH control. I n experiments 3, 4, 5, and 6, a 50-7 aliquot of calcium \vas niixecl with the appropriate volume of magnesium n-orhing solution and a precise volume of dilute sodium hydrovide solution. The mixture 11-as diluted t o 11.0 nil. n i t h water. Sodium hydroxide n a s utlded: 1.0 nil. of 0 . 2 5 for p H 11.8, 2.0 nil. of 0 . 2 s for pH 12.4 to 12.5, and 1.0 nil. of 2.0.1- for pH 12.8. As shovn in Table I a t pH 11.8, the presence of magnesiuni resulted in a
highrr 1-aluc for calcium; at pH 12.8, a l o ~ e rx-alue n-as obtained. Determination of the calcium at pH 12.4 to 12.5 was a useful compromise between these estrenies, magnesium interference being a t a niinimum. The regression line, I- = 100.71 - 0.171S, calculated from the pooled data of experiments 4 and 5 is plot'ted in Figure 1 together \\-ith the observed points. As interference is negligible (less than 2.0%) a t magnesium to calcium ratios up to 16, these conditions are used in the rrconimended procedure. DISCUSSION
K h e n determining calcium in the presence of a magnesium to calcium ratio of 0.4, normally encountered in human rind rat serum, the p H must be above 12, as suggested by ~ l u n s o nand his associat,es. At pH 11.8, this lonratio may result in a 3% error (Table I , experiment 3). In the presence of magnesiuni to c&iuiii ratios of 1 or grentrr or ~ h c nthe iiingiiesium content is unknown, the p H must be cont'rollril a t 12.4 to 12.5, L-nder these contlit'ions, no difficulty from magnesium interference should be encountered in the analysis of maiiinialian tissues and fluids. Nature of Magnesium Interference. T h e precise nature of t h e iiiechaiiisni of t'he interference b y magnesium is not known. T h e magnesium may interfere by foriiiiiig a complex eithrr \\-ith t h e aninioriium purpurate or with the EDTA. Combination with E D T A would teiid to increase the t,itratioii volume, resulting in a highcr value for calcium. Such a mechanism cannot be judged the major cause of magnesium interference in this study, \\-it11 the exception of the determinat,ions a t pH 11.8(Table I, experiment' 3) in which higher values were obtained. It is possible that a t lower p H values the magnesium interferenee results from competition with calcium for EDTA. Bond and Tucker ( 8 ) suggested this when describing magnesium interfcrence in a macromethod for calciuni, requiring up to 8000-ycalcium aliquots. At pH values of 12.5 and above, where t'lie calciuni in the presence of mngiiesiuni t'ends to be underestimated, another cause for the interference is sought. It has been suggested ( 2 ) that a t higher pH values magnesium interferes by adsorbing calciuiii on the magnesium hj-droxide precipitate. This is difficult to accept as the only factor in the present study, because increasing the purpurate to calcium ratio decreased the interference. Diehl and Ellingboe ( 3 ) r e p o r t d the successful analysis of calcium in the presence of magnesium to calcium ratios of 50 and aboi-e using a new indicator, calcein, with Ivliich magnesium does not form a complrs. VOL. 30, NO. 8, AUGUST 1 9 5 8
1367
Under the conditions of their titration, above p H 12, magnesium hydroxide would be present as a precipitate and available for adsorption of calcium and, according to the suggestion of Bond and Tucker ( 2 ) , would result in interference. As no interference occurred, it is unlikely that magnesium interferes by such a mechanism. The major interfering mechanism a t high p H might be the competition between calcium and magnesium for the ammonium purpurate. Should the magnesium be competing TVith calcium for purpurate complex formation, this interference would be negligible a t p H 11.3. At this pH, an isobestic point exists a t 505 mp ( 7 ) , where the absorption spectral curves for ammonium purpurate in the presence and absence of magnesium intersect. Tammelin and Rlogensen (6) reported a n isobestic point a t 510 mp a t a p H of about 11.
An experiment run a t 505 mp and p H 11.3using a 10-7calcium aliquot yielded means for four and three replicates, respectively, of 109.4 =k 2.47, and 110.8 =k 2.47, a t magnesium to calcium ratios of 5 and 10, respectively. Although under these conditions the competition of magnesium with purpurate may have been eliminated as a source of interference, the problem mas not solved. The interference caused by the competition of magnesium with EDTA remained. ACKNOWLEDGMENT
The authors wish to thank Oscar 4. Iseri and Paul L. Alunson for perniission to incorporate the details of their analytical procedure prior to full publication. They also wish to thank RIichaela Thompson for first drawing the question of magnesium interference to their attention, Mindel C. Sheps for
advice on the statistical aspects of the paper, and Virginia h'lellor for valuable technical assistance. LITERATURE CITED
( 1 ) Blaedel, IT. J., Knight, H. T., A s . 4 ~ . CHEL 26, 743 (1954). ( 2 ) Bond, R. D., Tucker, B. hZ., Chem. Le. Ind. (London) 1954, 1236. (3) ~, Diehl. H.. Ellinrboe. J. L.. ANAL. &EN. 28. 882 T1956). Kenny, -1.D Toverud, S. U.,Zbid., 26,1059 (19b4).
MLinson, P. L., Iseri, 0. A., Kenny, A. D., Colin, V., Sheps, 11. C., J . Dental Research 34, 714 (1955). Tanimelin. L. E.. Mogensen. S..' Acta Chem. Scand: 6, 98g (1952). Williams, 11.B., Moser, J. H., ASAL. CHEM. 25, 1414 (1953).
RECEIVED for review September 12, 1958. Accepted April 9, 1958. Supported in part by research grant D-203 from the National Institute of Dental Research of the National Institutes of Health, U. S. Public Health Service.
Radiochemical Determination of Neptunium-239 and Plutonium-239 in Homogeneous Reactor Fuel and Blanket Solutions FLETCHER L. MOORE Oak Ridge National Laboratory, Oak Ridge, Tenn. ,The radiochemical determination of neptunium-239 and plutonium-239 in uranyl sulfate fuel and blanket solutions is based on the carrying of these two isotopes by lanthanum fluoride followed by liquid-liquid extraction of the neptunium-239 with 2-thenoyltrifluoroacetone-xylene. Use of a single container for all separations minimizes losses.
T
HE radiochemical determination of neptunium-239 and plutonium-239 is required in connection rvith the homogeneous reactor program to determine the physical and chemical behavior of these elements in uranyl sulfate fuel and blanket solutions under pile irradiation. Previous radiochemical methods for the determination of neptunium239 (3) and plutonium-239 (4) were developed for application to nitrate or chloride systems, with free sulfate being a serious interference. Although the lanthanum fluoride technique ( 1 ) may be applied to solutions containing free sulfuric acid, the separation of neptunium and plutonium is inadequate. This paper describes the recently de-
1368
ANALYTICAL CHEMISTRY
veloped methods for the radiocheniical determination of neptunium-239 and plutonium-239 in pile-irradiated uranyl sulfate fuel and blanket solutions. These methods combine the better features of previous techniques (1, 3, 4). As depleted uranium was used, essentially all gamma radioactivity was due to neptunium439 nhich decays with a 2.33-day half life t o the alpha emitter, plutonium-239. The methods are based on the carrying of neptunium-239 and plutoniuni239 on lanthanum fluoride (1). dissolution of the precipitate, and liquidliquid extraction of the neptunium-239 Fvith 2-thenoyltrifluoroacetone-xylene. 411 precipitation and extraction steps are performed in the same centrifuge cone to minimize losses. A high-speed motor stirrer (Palo Laboratory Supplies, S e w York, N.Y.) with a glass paddle gives excellent mixing of the phases. NEPTUNIUM-239
Prepare necessary dilutions in 231 nitric acid. If a yield correction is to be applied, pipet a known amount of neptunium-237 in 2111 nitric acid solution into a 1% or 15-ml. Pyrex centri-
fuge cone (Corning No. S120 or 8060, respectively). Add a suitable aliquot of the sample dilution. Add 0.4 ml. of concentrated hydrochloric acid, 0.1 ml. of zirconium holdback carrier (10 mg. per ml.), 0.1 nil. of lanthanum carrier (5 mg. per nil.), and mix well. Add 0.3 ml. of 531 hydroxylamine hydrochloride and 0.3 ml. of 27W hydrofluoric acid. Stir well with a platinum stirrer. and digest for 5 minutes a t room temperature. Centrifuge for 3 minutes in a clinical centrifuge. Add 0.05 ml. of lanthanum carrier and stir the supernatant, being careful not to disturb the precipitate. Digest for 5 minutes a t room temperature. Centrifuge for 3 minutes, and carefully remove the supernatant b y use of a transfer pipet that is attached to a vacuum trap. Wash the precipitate by stirring 11ith a 0.5-nil. portion of 1-11 nitric acid-Llf hydrofluoric acid. Centrifuge for 3 minutes and carefully remove the supernatant, leaving approximately 1drop. Dissolve the lanthanum fluoride precipitate by adding 0.2 ml. of 2 X aluminum nitrate solution and 1 ml. of 4;M hydrochloric acid. Add 0.8 ml of 5 V hydroxylamine hydrochloride and 0.5 nil. of 231 ferrous chloride, freshly prepared in 0.2X hydrochloric acid every 2 Teeks. Adjust to approxi-
