Estimation of Submicroquantities of Calcium ALBERT E. SOBEL AND I. ALLAN KAYE The Jewish Hospital of Brooklyn, Brooklyn, N. Y.
Q
of the usual oxidimetric standards is unstable at, 0.01 9 strength and requires frequent checking. The use of a t least one stable standard in the titration is of Considerable advantage to the analyst who is often pressed for time. After establishing the validity of determining emall amount's of standard acid by Reactions 1 and 2 , a similar procedure mas applied to the microestimation of calcium in the calcium carbonate precipitate. The precipitate was dissolved in a n excess of 0.01 N hydrochloric acid. The excess acid was then determined by the addition of excess potassium iodate and potassium iodide and the liberated iodine back-titrated with approximately 0.0007 N sodium thiosulfate. It v a s possihle to determine with a high degree of precision amounts as low as 0.004 mg. of calcium, using a 5-cc. microhuret.
C A T T I T A T I V E isolation of small amounts of calcium may be achieved: (1) by precipitating the oxalate in a small volume, (2) by washing with small volumes of washing fluid, and (3) by employing a common ion-namely, oxalatein the washing fluid (IO). I n titrating the precipitate, two choices are available. First, smaller amounts of t h e titrating solutions may be used, in which case a specially designed capillary microburet is employed (IO). The second approach involves the use of more is dilute standard solutions-i. e., 0.001 to 0.0005 h'-and preferred in most laboratories because the capillary microburet is a t present not generally available and its use involves special care in calibration and manipulation. The main drawback in t h e use of very dilute standard solutions lies in the difficulty of obtaining a sharp end point. Bfter considerable investigation it was found that under controlled conditions the starch-iodide end point can be reliably employed with solutions as dilute as 0.0005 12' sodium thiosulfate; it was then possible to attack the problem of estimating submicroquantities of calcium with the ordinary microburet (5- to 10-cc. volume). An attempt was made t o determine calcium as t h e carbonate rather than the oxalate, as conversion of the calcium oxalate precipitate to calcium carbonate offers sereral theoretical and practical advantages over the direct use of calcium oxalate (4). T h e precipitate can be washed with ammonium oxalate, minimizing the loss of calcium oxalate through solution. Adsorbed oxalate ions, as well as coprecipitated ammonium oxalate, are volatilized during the ignition when the calcium oxalate is converted to calcium carbonate (or calcium oxide). Fewer washings are necessary. However, all the hitherto available microprocedures which employ calcium carbonate are acidimetric ( 3 , 5 - 7 , 9 , 11) and thus not suitable for a n iodometric titration, which is oxidimetric. It was possible, hon-ever, to convert a n essentially acidimetric procedure to an oxidimetric process by the following reactions: 2H+
+ KIO, (excess) + 5KI (excess) -+- + H20 +... I2 + 2Na2S203starch+ + 2SaI I2
6
Method REAGESTS. Hydrochloric acid, 0.01 'Y. Bromothymol blue, 0.04 per cent, prepared according t o Clark (1). Ammonium oxalate. A saturated solution of c . P. ammonium oxalate is prepared at about. 50" C. and allowed to cool to room temperature. The clear supernatant solution is used. Ammonium oxalate, 0.5 per cent, prepared by dissolving 0.5 gram of c . P. ammonium oxalate in distilled water and diluting to 100 ml. Sodium thiosulfate, 0.1 Ai, prepared according t o Kolthoff and Sandell (4). Sodium thiosulfate, 0.0007 to 0.0006 X , prepared by diluting 0.7 cc. of the 0.1 N sodium thiosulfate solution to 100 ml. with distilled water. This solution deteriorates very slowly on standing at room temperature-1 to 2 per cent in the course of a month. Potassium iodate, 0.05 per cent. Potassium iodide, 1 per cent. These solutions may be prepared roughly by the addition of a knife-tip of the solids, potassium iodide and potassium iodate, to a small amount of water. They should be tested before use for the presence of free iodine by mixing equal volumes with a few drops of starch. If a blue color develops, new solutions should be prepared. Starch, 0.2 per cent, prepared according to Iiolthoff and Sandell (4). APPARATCS. A specially designed Pyrex glass tube with wall 1.2 t o 1.5 mm. thick is used as a centrifuge tube. The over-all length is 74 to 76 mm. and the internal diameter is from 11 to 13 mm. The conical portion of the tube is about 23 to 25 mm. and it has a flat bottom approximately 2 t o 2.5 mm. in diameter. The flat bottom is important, as it permits the precipitate to pack down in a thin flat layer, easily dissolved subsequently, and it permits aspiration of almost the entire supernatant liquid above the precipitate. -45- or 10-ml. buret is calibrated to 0.01 ml. The 0.2-ml. pipet is a capillary pipet graduated in 0.01 ml. between marks. The over-all length of the graduated portion is about 140 to 160 mm. Glass stirring rods 3 t,o 4 mm. in diameter have an over-all length of 105 to 110 mm. One end is drawn out about 10 mm. to form a flat tip about 1 mm. in diameter. PROCEDURE. Into one of the specially designed Pyrex centrifuge tubes 0.2 ml. of solution is measured. (The tube should be
(1) (2)
I n the presence of a n excess of potassium iodate and potassium iodide the amount of iodine liberated is equivalent to the acid present, since Reaction 1 goes to completion (R, 4). The liberated iodine can be quantitatively titrated with standard thiosulfate in the presence of starch. Thus the amount of acid present in a given solution may be determined oxidimetrically by the sensitive iodine-starch end point. The titer of t h e relatively unstable sodium thiosulfate may be standardized by 0.01 h' acid whose titer is constant, whereas the titer 118
ANALYTICAL EDITION
FEBRUARY 15, 1940
cleaned with cleaning mixture before being used, thoroughly rinsed with distilled water, and then dried in an oven.) To this is added 0.2 ml. of saturated ammonium oxalate solution in such a fashion that the walls of the centrifuge tube are washed. One drop of bromothymol blue solution is then added and the contents of the tube are thoroughly mixed. The pH is adjusted to the faint blue color which corresponds to a pH of about 3.5. After standing at least 1 hour, the tube is centrifuged at 3000 r. p. m. for 15 minutes, and the supernatant liquid is aspirated with a drawn-out tube and rubber bulb without disturbing the precipitate. The precipitate is then suspended in 0.3 ml. of 0.5 per cent ammonium oxalate solution and recentrifuged at 3000 r. p. m. for 15 minutes. The supernatant liquid is removed by aspiration and the remaining liquid in the tube evaporated in an oven at about 110" C. When dry, the tube is placed in a muffle furnace a t about 560" C. for 1hour. After cooling to room temperature 0.2 ml. of 0.01 LV hydrochloric acid is added and the precipitate is broken up with a stirring rod. Solution is completed and the last trace of carbon dioxide expelled by placing the tube in a boiling water bath for about 15 minutes with occasional stirring. After cooling, 1 drop of 0.05 per cent potassium iodate is added, followed by 1 drop of 1 per cent potassium iodide. The contents of the tube are mixed thoroughly with the aid of the stirring rod and the amount of iodine liberated is titrated with an approximately 0.0007 N sodium thiosulfate solution. When most, of the iodine color has disappeared a few drops of 0.2 per cent starch solution are added and the contents of the tube are again mixed with the aid of the stirring rod. Titration is continued until the solution is colorless. This is easily visible in daylight against a white background. The sodium thiosulfate is standardized daily by titrating 0.2 nil. of 0.01 ,V hj-drochloric acid in the same fashion.
PROCEDURE FOR BLOODSERUM.The method is directly applicable to fresh serum except t h a t no adjustment of pH is necessary. At least 3 hours should be allowed for complete precipitation of t h e calcium oxalate. CALC~LATIOS. [Total ml. of 0.01 -I' HC1 - (ml. of r\'a2SnOs X factor)] X 0.2 = mg. of calcium ml. of 0.01 1V hydrochloric acid Factor = ml. of sodium thiosulfate equivalent to above aci4 mg. of calcium x 100 Mg. per cent of calcium = ml. of sample measured SAMPLE CALCULATION. 0.2 ml. of 0.01 N hydrochloric acid 3.10 ml. of sodium thiosulfate Factor =
0.2
-
3.10 Back-titration
hlg. of calcium
= =
1.53 ml. of sodium thiosulfate 0.2
-
0.02 Mg. per cent of calcium = loo = 10mg. per cent 0.2 since 0.2 ml. of sample was used originally for the calcium estima-
tion.
Discussion Calcium may be quantitatively precipitated as the oxalate
at a pH of 3.0 or higher (8, l a ) . A t a p H of 3.0 coprecipitation of magnesium is a t a minimum; therefore precipitation of calcium as t h e oxalate is recommended at a p H of 3.0 TI henever appreciable amounts of magneqium are present. T h e optimum temperature for t h e conversion of calcium oxalate to carbonate is between 475' and 525' C. (4). I n order to heat t h e calcium oxalate in this temperature range in a short time it is necessary to keep the temperature of the muffle furnace higher. T h e actual temperature of t h e muffle furnace in most of these experiments was between 560" and 600". With such a temperature the conversion of the oxalate to t h e carbonate is Completed in about 10 to 20 minutes. With a muffle furnace temperature of 490" to 530" this conversion takes a t least 3 hours. I n most of these experiments 1 hour a t 560 " to 600 " was employed, allowing a n ample margin of safety for complete conversion. Above 525" some calcium carbonate is decomposed to t h e oxide, but this does not
119
influence the results as t h e titrimetric equivalent of calcium oxide is identical with calcium carbonate. Hydrochloric rather than sulfuric acid must be used in dissolving t h e precipitate because of its difficult solubility in sulfuric acid. Solution is effected nithin 10 to 15 minutes with t h e aid of a stirring rod and immersion in a hoiling water bath. T h e heating, in addition to aiding solution, drives off dissolved carbon dioxide v hich M ould interfere v-ith t h e subsequent titration.
TABLEI. DETERMINATION OF CALCIUM IN IKORGANIC SOLUTIONS C a Present Mg.
0 0 0 0 0 0 0 0
C a Found M g
C a Present
0.003i 0 0040 0 0101 0 0102 0 0162 0 0162 0 0200 0 0200
0 0260 0 0260 0,0300 0.0300 0.0360 0 0360 0 0400 0 0400
.
0040 0040 0100 0100 0160 0160 0200 0200
Mo.
Ca Found Mg.
0 0263 p 025Y
0 0 0 0 0 0
0299 0298 0360 0353 0400 0393
It is important to have t h e solution a t loom temperature before adding potassium iodate and potassium iodide; otherwise volatilization of iodine may occur. The addition of the potassium iodate should precede the addition of potassium iodide for consistent results. The iodate and iodide solutions should be tested before use by mixing a few drops of each in t h e presence of starch. If this mixture shows a n y trace of blue color, new solutions must be prepared. These solutions need not be made up accurately. TABLE 11. DETERMINATION O F CALC1L.M IN SERUM Direct Acidimetric Method
I odometric Submicromethod
Mg./iOO ml.
If0 / l o 0 ml
9.8 6.9 9.7 10.5 10.0 9.5 8.8 11.0
9.8 6.9 9 5 10 1 9 8 9 4 8 6 10 8
T h e titration is best performed in daylight against a white background. Another centrifuge tube containing a n equal volume of distilled water is used to determine the water-clear end point b y color matching.
Results I n Table I representative results on known inorganic solutions containing 0.004 to 0.040 mg. of calcium are shown. The average deviation of t h e 16 consecutive estimations presented is *0.00018 mg. T h e ayerage percentage deviation in the same group is *1.2 per (Bent. The average error is -0.000056 mg. It is evident, therefore, that t h e present method is capable of a high degree of accuracy on knon 11 inorganic solutions. Comparative results on blood serum by the Sobel and Sklersky (9) direct acidimetric method and t h e present iodometric procedure are given in Table 11. I n the iodometric method 0.2 ml. of serum was used, whereas in the Sobel and Sklersky method 2.0 ml. of serum are employed. Excellent agreement is obtained 11y the two methods.
Summary A method is described for t h e titrimetric estimation of submicroquantities of calcium (0.004 to 0.0400 mg.) with t h e use of the ordinary 5- t o 10-cc. microburet. Calcium is precipitated as the oxalate in a specially designed centrifuge tube. T h e calcium oxalate is converted to the carbonate a t 475" to 525" C. T h e carbonate is dissolved in a n excess of 0.01 S hydrochloric acid by heating. T h e excess hydrochloric acid is determined iodometrically by the addi-
1NDUSTRIAL AKD ENGINEERISG CHEhllSTRY
120
tion of a n excess amount of potassium iodate and potassium iodide, which releases an amount of iodine equivalent t o the excess acid. T h e released iodine is determined by titrating with approximately 0.0007 S sodium thiosulfate, using starch as an indicator. This procedure retains the theoretical and practical advantages of the methods where calcium oxalate is converted t o calcium carbonate and combines with it the sensitivity of iodometric titrations. All solutions are standardized against the relatirely stable 0.01 S hydrochloric acid uqed.
Literature Cited (1)
Clark,
"Determination of Hydrogen Ions". 3rd ed., Baltimore. Williams d Wilkins. 1928 IT. M ,
(2) (3)
VOL. 12, NO. 2
Dumaaert, C.. Bzcll. SOC. chim. b i d . , 20, 1405 (1938). Fiske, C. H . , and .%dams. E. T.. J . A m . Chem. Soc., 53, 2498 (1931).
Kolthoff, I. h l . . and Sandell. E. B., "Texthook of Quantitative Inorganic Analysis", pp. 326-31, 589, 592, New York, Macmillan Co.. 1936. ( 5 ) Lebermann. F., .Uiinch. med. Tl'ochschr.,71, 1392 (1924) ; Chem. (4)
Zejatr.. 1, 139 (1925). (6) Nordbii, R., Riochem. Z., 246, 460 (1932). (7) Sieve, S.h., I h i d . . 278, 442 (1935). ( 8 ) Sobel. A. E., Pearl, A . , Gerchick, E., and Kramer. B.. J . Biol. Chem.. 118, 47 (1937). (91 Sohel. A. E., and Yklersky, S., Ibid., 122, 665 (1938). (10) Sobel, .1.E.. and Sohel, B. A , , I b i d . , 129, 721 (1939). (11) Trevan, J., and Bainhridge, H. W., Biochem. J . , 20, 423 (1926). (12) Washburn. 91. L . , and Shear, M. J., J . Biol. Chem., 99, 21 (1932).
An Improved Iodine Apparatus F. X. GASSNER Colorado Experiment Station, Colorado State College, Fort Collins, Colo.
F
OR the determination of traces of iodine in feedstuffs and similar material, the method of Karns (1) as modified by von Kolnit'z and Remington ( 2 ) seems to be the most
suitable procedure available from the standpoint of accuracy simplicity, and versatility. The metal torch designed tiy the lat8terauthors has certain disadrantages, and several changes have been made in the material, design, and construction of this apparatus which are heliered to he distinct improvements. To avoid the inconvenience of keeping the brass lacquered to prevent contamination of the sample by corrosion, the torch is constructed entirely of special resistant stainless steel, Allegheny chrome-nickel alloy 18-8. A further advantage of the stainless steel is its l o x heat conductivity. The four oxygen connectors are replaced by a single inlet tube, which supplies oxygen to the stainless steel jets through a built-in equalizing chamber. The outlet tube is also of stainless steel, since the glass tube sometimes cracked from the heat. All joints are soldered with special heat-resistant, corrosion-resistant material (special stainless steel solder furnished by ,J. T. Ryerson & Son, Inc., Chicago, Ill.). To eliminate the danger of explosion which occurred when cinders from high-ash samples clogged the constricted tube tip in the first Milligan wash bottle, a simple trap is attached in the manner shovn in the illustration. The prongs which hold the sample are made of Ferralloy, a material especially designed to resist oxidation a t high temperatures. The torch is reduced in length by 15 cm. (6 inches), which was found to be a distinct, advantage. It is, hon-ever, sufficientll- long to permit the burning of feed samples up to 30 cm. (12 inches) in length. The measurements used in construction of this torch conform otherwise to those given by von Kolnitz and Remington
(a).
The upper part of the torch is corered either bya 1000-cc. widemouthed ErIenmeyer flask or a 1-liter ivide-mouthed extraction flask with vial-type neck, The use of the latter flask resulted in less breakage and more rapid and more complete combustion. Discoloration of this part of the torch after long usage may be remedied by immersing it in a solution of concentrated nitric acid 25 per cent, hydrofluoric acid 3 per cent, and viater 72 per cent, and heating the mixture to '70" C. for 5 minutes. Care should be taken not to immerse the torch too far in the pickle. The solution must not come in contact with any soldered joint. T h e torch has also been found useful in determining the iodine content of biological material such as thyroid glands.
-4cknowledgment The changes herein described were made in collaboration with A. R. Patton.
Literature Cited (1) (2)
Karns, G. M . , IND. ENG.CHEY.,Anal. E d . , 4, 299-300 (1932). Kolnita, H. von, and Remington, R. E.. Ihid.. 5, 38-9 (1933).
