Spectrographic Analysis of Biological Material 11. Bismuth JACOB CHOLAK, Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio
of 450” to 500’ C. (Some difficulty due to fusing is encountered when materials rich in pho3phates are ashed. Such samples, especially liver and brain, are best ashed by a wet method employing sulfuric, nitric, and perchloric acids. If care is taken to destroy the char as soon as it is formed, bismuth will not be lost during the process of ashing.) The time required for complete ashing may be shortened considerably if the gray ash produced after a few hours in the furnace is treated with nitric acid, evaporated to dryness, and again placed in the furnace for about 15 minutes. The resulting white ash is dissolved in the smallest possible quantity of nitric acid and distilled TSater. The excess acid is neutralized with 20 per cent sodium hydroxide and the solution made just acid to methyl orange with (1 to 2) hydrochloric acid. An excess of 3 cc. of (1 to 2) hydrochloric acid for each 50 cc. of solution is added and the solution is treated with hydrogen sulfide for 1 hour. After it has been permitted to stand overnight in order to coa d a t e colloidal bismuth sulfide, the sulfide is filtered off on a dunktell No. 00 filter paper and washed with a hydrogen sulfide-saturated solution of 0.1 per cent hydrochloric acid. The sulfides are dissolved from the paper with hot (1 to 1)nitric acid and the paper is washed several times with hot distilled water. The filtrates containing the bismuth are then adjusted t.0 suitable volumes, as determined by the apparent quantity of sulfides obtained. Thus when a sample is small and when the sulfide is barelf visible, t’he filtrate may be concentrated conveniently to 2 cc., which may then contain up to 0.06 mg. of bismuth. When the sulfides indicate the presence of considerable quantities of bismut’h, any convenient concentration between 0.2 and 3.0 mg. of bismuth per 100 cc. may be employed. Two cubic centimeters of the properly adjusted bismuth solution and 2 cc. of a stock solution of inorganic salts containing 500 mg. of zinc per 100 cc. (the zinc line X 3036 serves as the “internal standard”) are mixed in a graduated 1.5-cc. Pyrex centrifuge tube and evaporated to 2 cc. in a water bath. [The concentrated stock salt solution used as diluent and as base for the standard bismuth solutions is made up by dissolving 170.5 grams of sodium chloride, 63.5 grams of potassium chloride, 31.5 grams of calcium chloride (CaCld3H20),20 grams of magnesium chloride (MgCla6H20), and 37.5 grams of sodium dihydrogen phosphate (NaH2POe,H20) in 1 liter of 10 per cent hydrochloric acid. Portions of this solution (50 cc., diluted to 100 CC. and made to contain 500 mg. of zinc) serve as the stock salt standard for mixing with t.he bismuth isolated from the samples and to make up the bismuth standards used to derive the calibration curves. This solu-
A method is reported for the spectrographic determination of bismuth in biological material. The method is capable of detecting 0.00004 mg. of bismuth and can be used to determine quantities from 0.001 to 3.00 mg. with an average error of * 10 per cent. SampGs as small as a fraction of a gram of tissue or 25 to 50 cc. of urine may be used.
T
HE quantities of bismuth present in biological material may be determined by a method similar to that described for lead (1, 2 ) . It is necessary first to isolate the bismuth in a concentrated form, since the most sensitive bis-
1,,,11
[ , ( I
1
,
,
(
,
1
010 015 0.20 PER lo0 CC. IxlcT4 .?*IOq4 3#d4 +&ON 7HE ARC BISMUTH CONCENTRATION (Me )
0.02 005
1.40 -
FIGURE 1
I
L
I ”
I
muth line (X3067.7) is partially masked by the iron line at X 3067.3 when material rich in iron is being analyzed, and since the presence of a faint narrow band in the spectrum of the graphite electrodes makes it impossible to detect bismuth unless the short-wave edge of the band is enhanced by having at least 0.00004 mg. of bismuth in the arc. Bismuth is not a common contaminant of reagents and as its sulfide is one of the least soluble of the heavy metals (solubility product = 3.2 X 4), its separation and concentration are not attended by great danger of contamination or loss. The disadvantage of increased manipulation is offset by the fact that the isolation of the bismuth permits the arcing of material identical in composition with the standards used to derive the working curves.
Technic
0.01 OD2 005 0.0 020 0.50 1.0 20 50 10.0 LO6 CONCENTRATION [He Bi PER loo LC)
The samples, digested with nitric acid and evaporated to dryness, are ashed in a muffle furnace maintained a t a temperature
FIGURE 2
26
JANUARY 15,1937
ANALYTICAL EDITION
27
I
with a sufficient quantity of the diluent to reduce the bismuth concentration. The alternative, when dealing with Bismuth added, quantities above 0.20 mg. of bismuth per 100 cc., is to deterBismuth mg. found, O.Ool O.O1 0 . 2 5 0.50 ‘.O0 2.00 mine the average density of the bismuth line corrected for mg. : 5::: : : : : variations in the density of the internal standard and to evaluate the bismuth concentration from the straight-line portion of Figure 2. These curves were plotted from the mean values TABLE11. &COVERY OF BISMUTH FROM TISSUESAND EXCRETA . of repeated observations on a series of solutions prepared by OF A RABBIT adding known quantities of bismuth and the required quantity (Killed one week after the administration of 3.5 mg. of bismuth of the “internal standard” to suitable volumes of the stock TABLEI. BISMUTH RECOVERIES
:
Tissue
:::
8 ii 8 i: !;
intravenously) Weight
Bismuth Found
Mo.
Gams
Blood Liver Spleen Kidneys Heart Lungs Central nervous system Testes Bile Pancreas Muscle Bone Skin Intestinal tract Contents of tract Ears, tail, and feet Remainder Entire carcass Urine Feces Total bismuth found in excreta Total bismuth found
::
76.1 54.8 0.7 11.5 4.1 6.2 14.0 1.4
Applicability, Sensitivity, and Accuracy of Method
0.001 0.013 0.000 0.08
0.000
0.001 0.000
... 2.1
0.000
578.5 132.0 208.4 132.2 164.0 130.0 348.0
0.014 0.18 0.01 0.05 0.03 0.10 0.05 0.633
o.ooo 0.004
~
’
2.24 0.90
-
3.140 __ 3.673
tion has proved very satisfactory, in that the salts present are good conductors and give a very steady arc when used with the technic described (1). Moreover it is free from the excessive creeping which may give rise to incrustations about the crater of the electrodes. Such incrustations result in spectrograms with very dark backgrounds because of the high concentration of incandescent salt particles in the arc.] Then a 0.2-cc. portion is introduced into each of 4 cratered graphite electrodes which are dried at 1200 e. and arced in accordance with the technic dewhile t,he region A2600 to A3800 is scribed for lead (I), photographed.
The recoveries for known amounts of bismuth added to digested portions of beef are tabulated in Table I. In Table I1 are recorded the results obtained from the tissues and excreta of an experimental animal. The weights of tissue available have been recorded in order to indicate the size of samples which can be utilized. The error of analysis, as shown in Table I, naturally depends on the quantity of bismuth encountered. For amounts above 0.20 mg. the error is = t l O per cent. For quantities from 0.01 to 0.20 mg. the error is about 10.01 mg. The greatest proportional error naturally occurs with quantities below 0.01 mg., but the errors in the estimation of extremely small amounts down to 0.001 mg. rarely exceed *20 per cent. (Although much smaller quantities can be detected, a t least as low as 0.00004 mg., quantitative estimations below 0.001 mg. do not appear sufficiently accurate to be useful because of mechanical losses, as those caused by adsorption, and the passage of colloidal bismuth sulfide through the filter paper.) I n the case .of material free from iron and containing very little ash, the separation of bismuth is of unnecessary, and it may well be that quantities as low as 0.0002 mg. of bismuth can be dealt with by further concentrating the solution of the ash,
Literature Cited Follo’ving the suggestion Of Lundegardh (’), two curves have been found useful in evaluating the densitometric values for the plates. The curve in Figure 1, in which the opacity ratios (galvanometer throw for the zinc line/galvanometer throw for the bismuth line) are plotted against the bismuth concentrations, is employed when the average opacity ratio of the four spectraindicates less than 0,0004 mg. (0.20 mg. per loo Of bismuth On the arc‘ This lnay be used in estimating higher amounts of bismuth by diluting
(1) Cholak,
J., IND, E ~ GCHEM,, , Anal, Ed,, 7, 287 (1935).
J., J . A ~Chem. , sot., 57, 104 (1935). (3) Lundegardh, H., “Die quantitat>ive Spektralanalyse der Elemente,” P a r t 11, Jena, G u s t a v Fischer, 1934. (4) Olseh, J. “Chemical Annual,” 7th ed., P. 477, NOW York, D. Van Nostrand Co., 1934.
( 2 ) Cholak,
c.9
RECEIVEDJuly 11, 1936. Presented before the Division of Physical and Inorganic Chemistry, Symposium on Spectroscopic Methods of Analysis, at the 92nd Meeting of the American Chemical Society, Pittsburgh, Pa., September 7 to 11, 1936.
Determining Calcium in Blood Serum H. K. MURER,’ Washington Agricultural Experiment Station, Pullman, Wash.
IN
place the funnel on top of test tube in the suction flask, dissolve the precipitate through the filter with three 2-ml. portions of hot N sulfuric acid, applying suction after each addition of acid, and fiTash the filters with water. &move and rinse the test tube and lower portion of filter into a 50-ml. beaker, heat to boiling, and titrate with dilute permanganate from a Koch automatic microburet.
THE determination of blood serum calcium by the Rymer and ~~~i~ (1) modification of the Tisdall (2) method, the separation and washing of the calcium oxalate by Successive centrifuging are very time-consuming. There is also possible loss while decanting and from creeping of the precipitate. By the use Of 30-cc*sintered-glass Buchner nation 3 G. 4),this centrifuging and decanting can be avoided.
Very satisfactory results were obtained with this method which lends itself well to fast routine work,
Place 2 ml. of serum in the test tube, dilute with 2 ml. of water and 0.5 ml. of saturated ammonium oxalate, shake, and allow to stand overnight. Filter on the glass filters, and wash with ammonia water (4 cc. of concentrated ammonia t o 250 ml.). After removing the funnel, rinse out the portion below the filter,
(1) Rymer, M.R., and Lewis, R. C., J . Bid. Chem., 95, 441 (1932). (2) Tisdall, F. F., Ibid.2 56, 439 (1923).
1
Present addresa, 44 Irving St., Cambridge, Mass.
Literature Cited
RECEIVED September 1, 1936.
