ANALYTICAL CHEMISTRY
1708 sample high in amine is added. If the basic color of methyl red appears during the determination, the sample should be discarded and a new determination should be started, using more acid to ensure an excess. Water, although a very weak base, if present in more than traces interferes seriously. Its weakly basic properties obscure the end point beyond detection. If the sample contains water in excess, it will separate out on cooling. In such a case, it is advisable to collect the sample in a thermos bottle. The water settles out readily and the sample can be decanted into the apparatus described. Acid gases do not interfere in this determination; hence it is not necessary to exclude carbon dioxide, hydrogen sulfide, or mercaptans. The method is useful for rapid control analysis. The equipment is easily portable for plant work. It is excellent for indication of trends and is time-saving where rapid analysis is required, The method is limited to gases that condense readily-i.e., propylene, propane, and higher. It is not suitable for referee work. The method of Donn and Levin answers admirably for these purposes. The accuracy of the titration is independent of the amine present. Qualitative investigation indicates the titrant is suitable for
titration of bases much weaker than ammonia and aliphatic amines. ACKNOWLEDGMENT
The author would like to acknowledge gratefully the assistance of some of his associates. Thanks are due to J. S. Frit,z and Ralph W. Sheets for their advice and encouragement throughout the work, and to Eugene S. Carlson for the drawing which appears in Figure 1. LITERATURE CITED
(1) Blumrich and Bandel, Angeu?. Chem., 54, 374 (1941). (2) Carlton, J . Am. Chem. SOC.,44, 1469 (1922). (3) Conant, Hall, and Werner, Ibid.. 49, 3047, 3062 (192;); 50, 2367 (1928); 52, 4436, 5115 (1930). (4) Donn and Levin, IND.ENG.CHEM.,ANAL.ED., 18, 693 (1946). (5) Ferner and Mellon, Ibid.,6, 345 (1934). (6) Fritz, ANAL. CHEM.,22, 578 (1950). (7) Ihid., p. 1028. (8) Kolthoff and Guss. J . Am. Chem. SOC.,60, 2516 (1938); 62, 249 (1940). (9) Michaelis and Mizutani, 2. physik. Chem., A116, 135 (1925). (10) Nadeau and Brancben, J . Am. Chem. SOC.,57, 1363 (1935). (11) Smith and Gets, IND.ENG.CREM.,AXAL.ED., 9, 378 (193;). (12) Wooten and Hammett, J . Am. Chem. Soc., 57, 2289 (1935). RECEIVED June 2, 1951.
Determination of Calcium in Biological Fluids I. J. GREENBLATT AND SEYMOUR HARTMAN' Beth-El Hospital, Brooklyn, N. Y . HE principle of Schwarzenbach and coworkers ( 3 - 5 ) in emTploying sequestering reagents in the presence of an indicator for the determination of alkali earth metals has long been known. It has been applied in the field of water analysis in the determination of water hardness, as described by Bet2 and No11 ( 1 ) . The authors have applied this principle for the determination of calcium in biological fluids. The calcium, in an ionic state, in the presence of an organic indicator produces a pink color ( 1 ) . When titrated with a sequestering reagent, the calcium is firmly bonded in an unionized soluble complex which turns orchid-purple.
the addition of another drop of sequestering reagent, will not alter. I n a similar manner a standard calcium solution (1 ml. = 0.1 mg. of calcium) is used, and the results are computed. I n comparison with the potassium permanganate methods ( 2 , 6), in which the calcium is precipitated as an oxalate, the results of the indicator method are in excellent agreement, as seen in Table I.
Table I.
Determination of Calcium ( h k . 7c)
APPARATUS AND REAGENTS
A casserole or an evaporating dish of 200-ml. capacity. A microburet of 1-ml. capacity. The organic indicator is prepared by mixing well 0.2 gram of ammonium purpurate with 100 grams of sodium chloride, and grinding the mixture to 40- to 50-mesh. Sodium Hydroxide, 1.0 AT. Titrating (Sequestering) Reagent. Disodium dihydrogen ethylenediamine tetraacetate dihydrate (4.0 grams) is dissolved in approximately 800 ml. of distilled water, 0.86 gram of sodium hydroxide is added, and adjusted against standard calcium chloride solution so that 1 ml. equals 1 mg. as calcium carbonate.
Blood Calcium
Urine Calcium
0.96 3 01 3.19 2.93 2.96 2 15 3.40
Present address, Children's Hospital, Akron, Ohio.
0.91 2.94 3.08 2.80 3.05 2.35 3.28
Spinal Fluid Calcium 5 0
1
10.4 9.05 9.06 9.08 9.33 11.4 8,15
10.7 9.18 9.4 9.08 9.33 11.4 8.2
PROCEDURE
A 0.5- to 2-ml. sample of a biological fluid (blood, urine, or spinal fluid) is pipetted into a casserole and 8 ml. of distilled water are added. The mixture is stirred rapidly with a glass rod (if necessary, a drop of caprylic alcohol may be added t o break any foaming), 2 ml. of 1 N sodium hydroxide are added to give the system the proper pH, and then one-third cup of indicator is added. [A calibrated cup (supplied with kit) holds approximately 0.21 gram of indicator.] At this time the solution is a salmonpink color. The mixture is then titrated with the sequestering reagent until a stable orchid-purple color is obtained, which, upon
Indicator
KMnOb
4 4 5 4
35 5 2 8
5.17 41 4.9 5.2 5.0
The advantages of the indicator method are that a determination is completed in approximately one tenth the time ordinarily required for a calcium determination in biological fluids. Furthermore, the results are in very good agreement with other methods for determining calcium (2, 6). The disadvantage and limitation of this method are encountered in its application to hemolytic and jaundice serums, because of the presence of high concentration of
V O L U M E 23, N O . 11, N O V E M B E R 1 9 5 1
1709
interfeiing substances. I n hemolytic serum in which there is a marked increase of iron ions, and in jaundice serum in which there is a marked increase in bile pigments-namely, bilirubin-these interfering substances (iron and bilirubin) cause a blurring of the sharp, distinct rnd point ordinarily obtained in titrating clear serun1s. ACKNOWLEDGMEYT
The authors wish to express their thanks t o 11. E. Gilwood for his s'@estions in this work' *I1 reagents were Obtained from W. €1. & L. D. Betz, Philadelphia, Pa.
LITERATURE CITED
J . -4m.Water W o r k s Assoc., 42, ( 1 ) Bets, J. D., and Noll. C. -I., 49-52 (1950). (2) Kramer, B., and Tisdall, T. F.,J . Bid. Chem.,47, 474 (1921). (3) Schwarzenbach, G., Helv. Chzrn. A c t a , 29, 1338 (1946). and Bangerter, F.. Ibid.. 29, (4) Schwarzenbach, G., Biedermann, W., 811 (1946). ( 5 ) Schwarsenbach, G., Kampetsch. E., and Steiner, R., Ihid., 28, 828 (1945). (, 6,) Tisdall, T. F., J . Biol. Chrrn.. 56, 439 (1923). RECEIVED March 30, 1951. Presented a t t h e ~Ieeting-in-Miniature,S e w York Section, S ERICA AS CHE\IICIL SOCIETY. Brooklyn, X. Y . . March 17. 1930.
Microdetection of Sulfur LEONARD P. PEPKOWITZ AND EDWIN L . SHIRLEY Knolls Atomic Power Laboratory, General Electric Co., Schenectady, !V. Y .
HE usual microdetection of sulfide depends on the reaction rbetween iodine and azide, which is catalyzed by sulfide ( 2 ) . For organic materials and other solids this requires a fusion with sodium or potassium metal that often introduces errors caused by the sulfur content of the alkali metal. .-i method for the microdetection of sulfate, also described by Feigl ($), depends on the permanent coloration of barium sulfate precipitated in the presence of potassium permanganate. The sensitivity of the procedure is given as 2.5 micrograms of sulfuric acid (W), but this pioredure is rrbtricted to sulfate ion in solution in the absence of 1e:id sulfate.
i 'i
onstrated that arsenic, antimony, selenium, and tellurium do not interfere, as they are not converted t o volatile reducing compdunds by the reducing mixture. Sitrate interferes by oxidizing the evolved hydrogen sulfide to elemental sulfur in the condenser and must therefore be removed. APPARATUS
The apparat,us, simply constructed from a length of 6-mm. borosilicate glass tubing, is indicated in Figure 1. The upper bulb is cooled with an air jet and the solution heat,ed with a microflame. The combustion tube, which is used for certain organic materials such as thiophene, is also a length of 6-mm. tubing with a short copper oxide plug held in place by glass wool. The combustion tube, when used, is joined to the still by a short length of Tygon tubing. Rubber tubing will produce erroneous results because of the sulfur content of rubber. The copper oxide is heated to a dull red with a small Bunsen burner. REAGEPITS
-,&--
ABSORPTION TEST TUBE
TYGON
-
/
Nz
Reducing Mixture. AIix 100 nil. of 4770 hydriodic acid, 160 nil. of concentrated hydrochloric acid, and 40 ml. of 30% hypophosphorous acid; boil for 20 to 30 minutes t o remove any sulfur contamination; cool and store for use. Molybdate-Thiocyanate Solution. Dissolve separately 1.25 grams of ammonium molybdate in 50 nil. of water and 2.5 grams of potassium thiocyanate in 45 ml. of water. Acidify each solution with 5 ml. of concentrated hydrochloric acid. Store the solutions separately in dropping bottles. Mix 0.5 ml. of each solution in the absorption test tube before each determination. PROCEDURE
Icm
WOOL
H SCALE
Figure 1 . Apparatus for Xiicrodetection of Sulfiir
The method described in this paper, while somewhat less sensitivr than the azide-iodine test, is applicable to sulfur in any form iiicludiiig sulfate, sulfide, or elemental sulfur. The procedure is I r : r d on the use of the reducing misture originally described tiy 1,uke (3)and used as the basis of a quantitative microprocedure by Pepkowitz (4,5). The reducing misture (HCI HI II.,POz)converts the sulfur in most materials directly to hydrogeri sulfide on gentle heating. This includes such substances as eleniental wlfur, barium sulfate, lead sulfatr, and a number of organic materials. Some organic substances, notably thiophene, niwt be pyrolyzed before reduction. The evolved hydrogen sulfide i j absorbed in an acidic ammonium molybdate-potassium thiocyanate solution ( I ). The niolyhdate is reduced and red molybdenum thiocyanate is formed. L-nder the conditions of the present test the procedure is sensitive t o 0.5 microgram of sulfur. Neither acetylene, nor sulfur dioxide produces the red coloration, hut the reducing misture will if allo\vrd to boil over into the receiver. I t u-as esperimentally dem-
+
+
hpprosimately 2 ml. of the reducing mixture are introduced into the still a t A and washed in with a drop of concentrated hydrochloric acid. This is easily done by tipping the still slightly. The nitrogen line is connected a t A and the gas is allowed to bubble through the still a t a slow rate so as not to carry over any of the reducing misture. The air jet is turned on and the reducing mixture is boiled gently with a microflame for 2 to 3 minutes t o remove any sulfur contamination. The absorption tube is not in position during this step in the procedure. The still is allowed to cool with the gas stream flowing while the absorbent is being prepared. When cool, the gas line is removed and the sample is introduced at A . Solid samples can be placed in A and washed into the still with a drop of concentrated hydrochloric acid or they can he introduced in a small glass capillary which is pushed into the tube and broken off a t the bend. Liquids are introduced by the same procedure. The gas line is replaced and the apparatus assembled with the distillation tube dipping into the absorbing solution. The gas flow is adjusted so as not to spatter the absorbent and the reducing solution is heated gently with a microflame to a gentle reflux. I n a feu- seconds the absorbent will turn red if any sulfur is present. For the more refractory organic materials, the reducing misture is intrcduced and the still assembled, including the combustion t,ube. The reducing mixture is purged as before and cooled with the gas flowing. When cool, t'he absorber is placed
