(5) Kirk, P. L. “Quantitative Ultramicroanalysis,” diley, New York, 1950. (6) Kolthoff, I. M., ANAL. CHEM. 26, 1685 (1954).
(7) Kolthoff,’I.M., Lingane, J. J., “Polarography,” 2nd ed., Vol. 11, p. 887, Interscience, New York, 1952. (.8.) Kolthoff, I. M., Steneer. V. il.. “Volumetric Analysis,” 2Gd ’ ed., Vol: I, Chap. VI, Interscience, New York, *n>n
lY4L.
(9) Lazarow, Arnold, J. Lab. Clin. M e d . 35, 870 (1950). (10) Levy, G. B., Murtaugh, J. J., Rosen-
blatt, Maurice, ISD.ENQ.CHEM.,ANAL, ED. 17, 193 (1945).
(11) Mitchell, John, Jr., E. I. du Pont de Nemours & Co., Inc., Wilmington
Del., private communication.
(12) Mitchell, John, Jr., Smith, D. M.,
“Aquametry,” Interscience, New York, 1948. (13) Neuss, J. D., O’Brien, M. G., Frediani, H. A., ANAL. CHEM. 23, 1332 (1951). 114) Peters. E. D.. Junenickel. J. L.. ‘ Ibid., 27,’450 (1955). (15) Potter, E. C., White, J. F., J. ’ A p p l . Chem. 7 , 309 (1957): (16) Roth, C. F., Mitchell, John, Jr., A N A L . CHEM. 28, 1502 (1956).
(17) Stock, J. T., Metallurgia 55, 48 (1957). (18) Stone, K. G., Scholten, H. G., ANAL. CHEM.24, 671 (1952). (19) Wernimont, Grant, Hopkinson, F. J., IND. ENQ.CHEM., ANAL. ED. 15, 272 (1943). (20) Wiberley, J. S., ANAL. CHEM. 23, 656 1951). (21) Jakubik, M. G., J . Chem. Educ. 35, 5(1958).
RECEIVED for review May 2, 1958. Accepted September 19, 1958. Division of Analytical Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958.
Microtitration of Calcium to Visible End Point in Presence of Magnesium Sodium (Ethylened initri1o)tetraaceta te as Titrant SIDNEY J. SOCOLAR’ and JAMES 1. SALACH College and Deparfmenf o f Physiology, The University o f Chicago, Chicago, 111. ,Calcium of the order of 1 y or less can b e determined by titrating to a visible end point with sodium (ethylenedinitrilo)tetraacetate, obviating the need for photometry. The procedure employs calcein indicator and is therefore applicable in the presence of magnesium. The standard deviation of an individual determination is.about 0.03 y of calcium.
S
procedures have been described for the determination of microgram and submicrogram quantities of calcium (4-6). All are photometric. One of these ( 4 ) is a n adaptation of the well known method wherein the end point of a titration with (ethylenedinitri1o)tetraacetic acid (EDTA) is indicated by murexide dye. The others (6, 6) employ organic dyes whose colors are sensitive functions of calcium ion concentration over a considerable range. Volumetric titration to a visible end point is often more convenient than photometry. Such a procedure has been developed and applied to quantities of calcium as small as 0.2 y . It appears to be simpler and, for many applications, more rapid than at least two of the previous methods (4, 6). Within broad limits it avoids the problem of magnesium interference. The procedure reported here employs calcein, the end point indicator described by Diehl and Ellingboe ( 2 ) . They noted that calcein in highly alkaEVERAL
Present address, 430 West 118th St., Kew York 27, K . Y.
line solution is normally brown, but assumes a yellow-green color in the presence of calcium ions, which jt complexes. At high p H calcein binds magnesium far less strongly than it binds calcium. [The color of the calceincalciuni complex has since been ascribed to fluorescence, largely absent a t high p H if the calcein is free ( 7 ) . ] The procedure described here does not discriminate between strontium or barium and calcium; hon-ever, interference by copper or iron is prevented by the addition of cyanide ( 2 ) . Neither sodium nor potassium interferes. APPARATUS AND REAGENTS
Titration Vessels. The titration is done in a 4-ml. white porcelain crucible (Coors No. 0000, high form). Crucibles used concurrently should not differ perceptibly in color, as a slight variation in whiteness of sample and blank crucibles interferes seriously with determination of the end point. Glassware Treatment. Crucibles and glassware, except for silicone-coated volumetric pipets (used for all but strongly alkaline solutions), are cleaned in the conventional manner, then soaked for 6 hours in 2N nitric acid, and rinsed with metal-free water until the effluent rinse is neutral to methyl red (1). Titration Light. The titration is done by the condensed light from a tungsten source passed through a triple thickness of Kodak Wratten filter No. 78 AA. The source used by the authors was the lamp-type G. E. G161/2-29 (100 watts, 120 volts), in a microscope illuminator. This light provides good contrast between the colors of the calcium-free
blank and the calcium-containing solution. Buret. A Gilmont ultramicroburet (Emil Greiner Co.) of the steel piston in mercury type was used. The instrument has a 0.1-ml. capacity and its dial is graduated in 0.1-pl. divisions. Magnetic stirring is provided during the titration. Water. Metal-free water is prepared by passing singly distilled water over Amberlite MB-3 (analytical grade) ion exchange resin. Titrating Solution. A solution 0.002M in EDTA is prepared from the disodium salt according to Buckley et al. (I) and standardized against Iceland spar. Hydroxide-Cyanide Solution. A single solution is prepared 1N in sodium hydroxide, 0.7N in potassium cyanide. Indicator. Calcein indicator is purchased in powder form from the G. Fredrick Smith Chemical Go., Columbus, Ohio. For use, 1 drop of a 2% stock solution (2) is diluted with water to 25 ml. Observed color changes show that the dissolved indicator is unstable in ordinary light; therefore, solutions are kept in lighttight (foil-covered) containers. This instability is more readily noticed in dilute solution. The titrant, 2% indicator stock solution, and hydroxide-cyanide solution are stored in polyethylene bottles. Iceland Spar. The Iceland spar used in this work was supplied by the J. T. Baker Chemical Co. and was designated “for standardizing.” PROCEDURE
Into a 4-ml. crucible are pipetted 0.5 ml. of sample, 0.3 ml. of hydroxidecyanide solution, and 0.5 ml. of diluted VOL. 31, NO. 3, MARCH 1959
0
473
indicator. A second crucible is prepared similarly, the 0.5 ml. of sample being substituted by an equal volume of water. During titration the crucibles are illuminated by the filter-equipped tungsten lamp; extraneous artificial light should be excluded. The calciumfree blank then appears pink and the calcium-containing solution greenish yellow. The sample is titrated with 0.002M EDTA while being stirred continuously. The end point is reached when the last trace of yellow is gone from the sample and the latter matches the blank in hue and saturation. The titration requires less than 5 minutes. When several aliquots of a particular specimen are to be titrated, all the sample crucibles are prepared a t the same time and are shielded from dust until used. A single blank suffices for such a batch of successive titrations. However, because the color of the indicator in the crucible deteriorates in time, a given blank preparation should not be used with samples prepared appreciably later than itself. RESULTS
A standard solution about 0.001M in calcium was prepared by dissolving Iceland spar in hydrochloric acid. A portion of this solution was then diluted to 0.0004M and used to standardize a 0.002M solution of EDTA. Measured volumes of the 0.0004M calcium solution were diluted four-, sixteen-, and fortyfold, respectively. Samples of the three resulting solutions were analyzed for calcium; the results are shown in Table I. This investigation n-as undertaken to facilitate calcium determinations in small quantities of biological material. Hence, a check was made of the recovery of a known increment of calcium introduced into a sample of biological origin. A 4-ml. quantity of a solution of dry-
Table 1.
Ca present, y Ca found, y
a
Ave,. Y Std. dev., y Std. dev., % Recovery, yo Calculated t,o be 1.014 y.
Results of Calcium Determinations
Std. Ca 1.966 1.95 1.93 1.89 1.87 1.94 1.92 0.034 1.7 97.5
Yerve Ash FTith Ca addeda
Solution Aliquots 0.494 0.198 0.49 0.21 0.44 0.22 0.49 0.20 0.51 0.28 0.50 0.20 0.49 0.22 0.027 0.033 5.5 17. 98.4 112.
ashed lobster nerves in dilute hydrochloric acid was treated with Amberlite IRA-401 ion exchange resin (acetate form) and then made up to 10 ml. with rinses of the resin. [This procedure, following in principle that of Hahn et al. (S), was explored briefly as a means of eliminating possibly troublesome phosphate from samples to be analyzed for calcium. However, it has not been established that phosphate interferes with the titration.] Four 0.5-ml. samples from this resin-treated solution were analyzed for calcium. To a second 4-ml. portion of the same nerve ash solution, 0.5 ml. of the nominal 0.001M standard calcium solution was added. This was also passed over Amberlite and analyzed in the same way as the first portion. The results of these tests are given in Table I. The recovery of the added 1.014 y of calcium was calculated to be lOO.S%, with a standard deviation of 0.043 y, or 5.2%. The standard deviation of an individual determination, calculated from the pooled data of Table I, is 0.032 y. MAGNESIUM INTERFERENCE
Magnesium did not interfere when 0.5-7 calcium aliquots were titrated in the above manner, even when a hundredfold molar excess of magnesium
...
...
4.04 4.10 4.03 4.07
5.03 5.13 5.07 5.10
4.06 0.032
5.08 0.043
...
...
...
...
wis present. Only when the magnesium content was so high as to give visible precipitates upon addition of hydroxide did recovery of calcium appear significantly reduced. While these cases were not probed further, it seems plausible that significant quantities of calcium might be occluded in such precipitates. ACKNOWLEDGMENT
The authors acknowledge gratefully the helpful interest of Julian M. Tobias and of Thomas B. Coolidge. LITERATURE CITED
(1) Buckley, E. S., Jr., Gibson, J. G., 11, Bortolotti, T. R., J . Lab. Clin. Med. 38,
761 (19511.
(2) Dikhl, H., Ellingboe, J. L., ASAL.
CHEW28, 882 (1956). (3) Hahn, R. B., Backer, C., Backer, R., -4nal. Chim. Acta 9, 223 (1953). (4) Keynes, R. D., Lewis, P. R., J . Physiol. (London) 134, 399 (1956). (5) Kingsley, G. R., Robnett, O., Am. J . Clzn. Pathol. 27, 223 (1957). (6) Natelson, S., Penniall, R., A S A L . CHEM.27, 434 (1955). (7) Tucker, B. M., Analyst 82, 284 (1957). RECEIVED for review July 9, 1958. - 4 ~ cepted October 21, 1958. Work supported in part by a grant from the United States Public Health Service and in part by a grant from the Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago t o Julian M. Tobias.
Solubility of Cesium Metaperiodate and its Coprecipitation in the Determination of Potassium as the Metaperiodate SIR: Recently Jentoft and Robinson (2) reported the determination of potassium by precipitation of potassium metaperiodate from a 3301, (by volume) ethyl alcohol solution buffered to p H 3.5 with lithium acetate, followed by iodometric estimation. The present authors have investigated the possible application of this method to the determination of cesium, because no very successful method is available a t present. Measurement under the conditions demanded by this method 474
0
ANALYTICAL CHEMISTRY
has shown that the solubility of cesium metaperiodate is too large for a successful determination. Following the procedure of Jentoft and Robinson (1) it was established the minimum solubility occurs in a 20y0 ethyl alcohol medium. In such a solution 2.79 mg. of cesium as the metaperiodate are soluble in 5 ml. of solution a t 0" C. and buffered to pH 3.5. Further details may be found in a thesis ( 3 ) . The degree of the interference of cesium in the determination of potas-
sium by the metaperiodate method has not been previously ascertained. Potassium solutions containing cesium in varying amounts were analyzed by the method of Jentoft and Robinson. Samples containing I, 5, 10, and 25 nig. of potassium were prepared with 1, 2.5, and 5 mg. of cesium present for each concentration level of potassium. Solutions containing 10 mg. or less of potassium were also analyzed with only 0.5 mg. of cesium present. I n earlier experimental work on the solubility of cesium
