V O L U M E 20, NO. 4, A P R I L 1 9 4 8 the use of a number of receiver assemblies in case more than four fract,ions were desired. %lOLECULARSTILL
The receiver of the molecular still (Figure 4) is identical with t h a t of the microstill. The distillate is collected upon the flanged section of the straight, inclined cold-finger condenser and transferred to the receivers with the aid of capillary tubes 0.2 mm. in inside diameter. I n operation, the boiler, containing a 50 to 150-111. charge, is inserted into the cavity of the heating block clamped with its long axis horizontal. At the pressure obtainable with a diffusion pump, or v i t h a good mechanical pump, high boiling oils-e.g., dibutyl phthalat,e-could be distilled a t a rat,e of 20 pl. per minute at temperatures belmv 50' C. when ice water was passed through ?he condenser. LITERATURE CITED
(1) Baker, R. H.. Barlieribus, C., and Roswell, C. A , ISD. ESG. C H E M . . .4X.\L.
En., 12,408 (1940).
363 ( 2 ) Bower, J. R., Jr., and Cooke, L.
M,, Ibid., 15, 290 (1943). (3) Cheronis, N. D., and Levin, N..J . Chem. Education, 22, 86 (1945). (4) Craig, L., IND.ENG.CHEY.,ANAL.E D . ,9, 441 (1937). (5) Lecky, H. S., and Ewell, R. H., Ibid., 12, 544 (1940). (6) Morton, A. A., and Mahoney, J. F., Ibid.,l3,494 (1941). (7) Naragon, E. A., and Lewis, C. J.,Ibid., 18,448 (1946). (8) Rosanoff, M. A., and Easley, C. IY., J . Am. Chem. SOC., 31, 953 (1909). (9) Selker, M. L., Burk, R. E., and Lankelma, H. P., IND. ENG. CHEW,AXAL.ED., 12, 352 (1940). (10) Shrader, S. A , , and Ritzer, J. E., Ibid., 11, 54 (1939). (11) U. S. Dept. Commerce, OTS,PB 5951 (1946). (12) Weston, P. E . , IND.ENG.CHEM., AXAL.E D . ,5, 179 (1933). RECEIVED October 27, 1947. Contribution 1155 from the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, Calif. Based upon work done for Office of Scientific Research and Development under Contract OEhIsr-325 with the California Institute of Teehnology.
Microdetermination of Bismuth in Biological Material A n Improved Photometric Dithizone Method DOS.ILD 31. HUBBARD Ketterirag Laboratory of .4pplied Physiology, College of .I.ledicine, Unicersity of Cincinnati, Cincinnati, Ohio . i n improved photometric dithizone method has been developed for the determination of bismuth in biological material, applicable to 100 ml. or less of urine and 20 grams or less of blood. Bismuth is originally isolated from the highly diluted digested material, using a solution of dithizone in chloroform (60 nig. per liter), lead is separated with a buffer solution of pH 3.4, and bismuth is finally determined
A
N IAIPKOVED photometric dithizone method for the determination of bismuth in biological material has been developed recently and applied in this laboratory. Following preparation of samples by the regular n-et-ashing procedure ( 5 ) ,bismuth need not be isolated from mixtures of extraneous salts as the sulfide ( 4 ) , but can be extracted directly and qua,ntitatively as bismuth dithizonate. Separation from the lead dithizonate extracted simultaneously is effected a t pH 3.4, as indicated by Bambach and Burkey ( I ) , the final estimation of bismuth being made a t high pH, as described by Snyder (6). The sensitivity of the procedure is equivalent to that of the sulfide separation (4). REAGENTS AND APPARATUS
Ordinary high-grade chemicals m3y be used throughout the method. Eastnian dithizone may be used without further purification. The chloroform used may be a freshly obtained product after redistillation from Pyrex or used chloroform reclaimed by a procedure previously described ( 1 ) . All Squibb-type (PJ-rex) separatory funnels used must be cleaned meticulously with hot 50uc by volume nitric acid and distilled water to ensure removal of any bismuth or lead present as surface contaminntion from previous use ( 4 ) . The same applies t o the matched pairs of cells used for density measurements. Any standard spectrophotometer ma>- be used for nieasurenients of density. Instruments must be capable of isolating a narrow band centered a t 505 mM. PROCEDURE
Preparation of Samples. Samples of biological material, 100 nil. or less of urine, or 20 grams or less of blood or tissue, are prepared for analysis by a n-et-ashing method ( 5 ) . The digestion is made in a distilling flask, 1 liter in volume, provided with three necks possessed of interchangeable ground-glass connections, into
spectrophotometrically. The accurac? is within *0.1 microgram of bismuth for the range 0 to 10 micrograms and *0.5 microgram for the range 0 to 50 micrograms. Only one strength of dithizone solution is used throughout the complete procedure. Although used specifically for urine and blood, the method can be applied to other biological material. Bone samples may require special treatment.
n-hich are fitted the distilling head and txvo separatory funnels. This method of digestion involves the addition of 20 ml. of concentrated sulfuric acid (specific gravity 1.84), 5 ml. of perchloric acid (70 to 727,), and 20 ml. or more of nitric acid (specific gravity 1.42). The di ested sample is allov-ed to cool, 50 ml. of distilled water are afded, and the solution is transferred quantitatively to a 400-ml. Pyrex beaker. The beaker and contents are placed in a n ice water bath, 15 ml. of 407, w j v ammonium citrate solution and 50 ml. of 20% TV/V sodium sulfite solution are added; then, with stirring, 100 ml. of ammonium hydroxide (specific gravity 0.9) are also added. Extraction 1. The prepared sample is transferred quantitatively to a 500-ml. Squibb-type separatory funnel. After the addition of 5 ml. of 107, w/v potassium cyanide solution, the mixture is diluted to 400 ml. with distilled water and mixed thoroughly. Bismuth and lead are extracted as the dithizonates by successive additions of 10-ml. portions of dithizone in chloroform (60 nig. per liter) until the last portion shows no color change. For amounts of bismuth not exceeding 10 micrograms, three portions of dithizone solution suffice. If the amount of bismuth exceeds 10 micrograms but is not over 50 micrograms, four portions of dit,hizone solution are necessary. Each additional 10-ml. portion is roughly equivalent to 50 micrograms of bismuth. The combined extract is collected in a 125-m1., graduated, Squibbtype separatory funnel. Depending upon the amount of bismuth present, either the whole or an aliquot of the total previous estract, containing not more than 50 micrograms of bismuth, is taken for Extraction 2, and washed wit,h 50 nil. of distilled mater. after separation of the tn-o phases, t,he chloroform phase is transferred to a clean separatory funnel and the aqueous phase is shaken with 5 ml. of dithizone in Chloroform (60 mg. per liter); the latter is added to the chloroform phase but the \\-ash water is discarded. Extraction 2 'Removal of Lead.) The chloroform phase containing bismuth and lead dithizonates plus excess dithizone is shaken for 30 seconds with 50 ml. of buffer solution, pH 3.4 (I), and the chloroform phase is transferred to a clean separatory
ANALYTICAL CHEMISTRY
364 Table I.
.4nalysis of Urine
Range Used, Microgram
Bismuth Added, Micrograms
0-10 0-10 0-10 0-10 0-10 0-10 0-10 0-10 0-50 0-50 0-50 0-50 0-50 0-50 0-50
0 0
0-50
1
5
10 10 10 10 30
30 50 50 500 500
Bisniuth Found, Micrograms 0.1 Nil
0.9 1. o
5.0 5.1 10.1
9.9 10.5 10.0 30.0
29.5 50 50
505
495
funnel. (Separatory funnels used must be scrupulously clean.) The buffer solution is shaken with an additional 5 ml. of dithizone in chloroform (60 mg. per liter) and this is added to thr main chloroform fraction. The aqueous buffer solution containing lead is discarded. (In the presence of large amounts of lead, it will be necessary to repeat the above treatment in order to make certain that all of the lead has been removed.) The conibined lead-free chloroform fraction is next shaken for 30 seconds with 25 ml. of 1% v/v nitric acid and the chloroform fraction is removed to another clean separatory funnel. The treatment with 25 ml. of 1% v/v nitric acid is repeated, the acid fractions being combined while the chloroform fraction is discarded. The combined acid phase is washed with 5 ml. of clear chloroform to remove traces of dithizone, and all the excess chloroform is drawn off. (Care must be taken not to allom- the dilute acid phase to enter the bore of the stopcock.) The bismuth is now present in 50 nil. of 1% v/v nitric acid. Extraction 3 (Estimation of Bismuth). Twenty milliliters of ammonium hydroxide (specific gravity 0.9) containing 0.2 gram of potassium cyanide (10 grams of potassium cyanide per liter of ammonium hydroxide w/v) and 15 ml. of dithizone in chloroform (60 mg. per liter) are added and the mixture is shaken for one minute. A small pledget of cotton is placed in the tip of the separatory funnel stem, and 5 ml. of the chloroform phase are filtered through and discarded. The proper sized cell is then filled with the filtered chloroform layer and its optical density (or transmittancy) is obtained a t 505 mp. A blank consisting of 50 ml. of 1 9 v/v nitric acid is also treated as in Extraction 3 in order to determine the zero bismuth reading. (One blank is sufficient for a series of samples dealt with on any one day.) The readings are then evaluated from charts prepared beforehand from known amounts of bismuth treated as in Extraction 3. Two ranges are used, 0 to 10 micrograms and 0 to 50 micrograms, depending on the cell lengths. One calibration chart is derived for each range. The zero in each case is used to determine the chart zero and readings are evaluated from a fresh curve drawn parallel to the original calibrated curve, passing through the zero of the blank. The cells used are cylindrical, having optically plane ends and an internal diameter of 14 mm. These cells must he metirulously cleaned and dried before use. AhALYTICAL RESULTS
111Table I are listed results obtained by the analysis of 100-ml urine samples (in duplicate) containing known added quantities of bismuth. I n ?‘Pble I1 are listed results obtained by the analysis of 20-gram samples of rabbit blood (in duplicate) containing knrian added quantities of bismuth. DlSCUSSIOh
Stitniious tin arid monovalent thalliuni do not interfere anti lead is removed during Extraction 2 with the buffer solution, pH 3.4. Stannous tin as such is oxidized during the sample preparation and therefore it does not interfere ( 3 ) . The same holds for thallium (I). Thallium (111)is not extracted during the initial extraction step which involves a pH of approximately 10. Should thallium (I) by chance avoid oxidation and thus be extracted during the initial extraction step, it would be subsequently removed a t pH 3.4 during the removal of lead. Bismuth is quantitatively extracted (Extmrtion 1) evrn in the
presence of aniiiioniuni citrate, calcium, ferric, and phosphate ions, and chloride and other extraneous ions present in the original digest, because of a high dilution factor, a high pH of approximately 10, and a relatively strong solution of dithizone in chloroform. In dealing with minute quantities of bisniuth in the presence of unusually large quantities of lead (Extraction 2), one may find that the bismuth cannot be shaken completely into 1% v/v nitric acid because the great excess of free dithizone liberated from the lead dithizonate during the pH 3.4 wash tends to hold bismuth in the chloroform phase. However, if a stronger acid solution, such as 5 % v/v nitric acid, is used, all the bismuth will strip into the stronger acid. When such samples are encountered, it will be necessary to re-extract the bismuth, including the pH 3.4 wash, before proceeding to the final colorimetric extraction. For samples examined by this laboratory, such a step has not been required, but it is given as a precautionary procedure for determining small amounts of bismuth in the presence of large amount& of lead. Table I I. tlaiige I‘aed.
\Iirrogram\ 0-10 0-10 0-10 0-10
0-10 0-10 0-10 0-10
0-50 0-50 0 -50 0-50 0-50 0-50 0-50 0-50
inalFsis of Blood
B1umut.h -4dded \Iicrogram0
n 1 1 5
5
in
in 10 10
30 30 R0
50 500 500
Bismuth Found, Slicrograms Xi1 Xi1 1.0
0.9 5.0
1.9 9.8 10.0 10.5 10.0
30.5 30.5 50
50 505 300
For F:utrttction 3, one strength of dithizone in chloroform (60 nig. pel liter) is used for both ranges-namely, 0 to 10 micrograms arid 0 to 50 micrograms of bismuth. The high pH of tipproximately 11.5 obtained in Extraction 3 permits the removal of the major part of the excess dithizone to the aqueous phase, and, in effect, ammonium dithizonate is actually used for the quantitative extraction of bismuth. It has been found convenient to store the solution of potassium cyanide in ammonium hydroxide (IO g r a m of potassium thiocyanate per liter of ammonium hydroxide w/v) in a refrigerator. The sensitivity of extraction 3 is *0.1 microgram of bismuth for the range 0 to 10 micrograms and *0.5 microgram for the range 0 to 50 rnicrograms. Although used specifically for urine and blood, the method van be readily applied to other biological material. Some difficulty ma> be encountered in analyzing bone, but small samples may be analyzed, provided the phosphate content is not greater than that normally found in 100 ml. of urine. Larger bone samples are best handled by introducini an initial step of bismuth separation as the sulfide, solution of the sulfide in nitric acid (2, d ) , and subsequent treatment as outlined above, starting with ‘Weparat ion of Samples.”
,
LITERATURE CITED
(1) Bairibach, K., a n d Rurkey, R. E., IND.ENQ.CHEM.,ANAL.ED., 14, 904 (1942). (2) Cholak, J., Ibid., 9, 26 (1937). (3) Hubbard, D. M.,I b i d . , 9 , 493 (1937). (4) Ibid., 11, 343 (1939). (5) Ibid., 13, 915 (1941). (6) S n y d e r , L. J., ANAL.CHEM.,19, 684 (1947). RECEIVEDOctober 11, 1947. Presented before t h e Division of Analytioal and Micro Chemistry at the 112th hleeting of t h e AMERICANCHEMICAL SOCIETY, ?iew York. N Y .
