Spectrographic Analysis of Biological Material

11. Bismuth
JACOB CHOLAK, Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio

A method is reported for the spectro- of 450” to 500’ C. (Some difficulty due to fusing is encountered
when materials rich in pho3phates are ashed. Such samples,
graphic determination of bismuth in bio- especially liver and brain, are best ashed by a wet method employ-
logical material. The method is capable of ing sulfuric, nitric, and perchloric acids. If care is taken to
destroy the char as soon as it is formed, bismuth will not be lost
detecting 0.00004 mg. of bismuth and can during the process of ashing.) The time required for complete
be used to determine quantities from 0.001 ashing may be shortened considerably if the gray ash produced
after a few hours in the furnace is treated with nitric acid, evapo-
to 3.00 mg. with an average error of * 10 per rated to dryness, and again placed in the furnace for about 15
cent. SampGs as small as a fraction of a minutes. The resulting white ash is dissolved in the smallest
possible quantity of nitric acid and distilled TSater. The excess
gram of tissue or 25 to 50 cc. of urine may be acid is neutralized with 20 per cent sodium hydroxide and the
used. solution made just acid to methyl orange with (1 to 2) hydro-
chloric 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
T HE quantities of bismuth present in biological material
may be determined by a method similar to that de-
scribed for lead (1, 2 ) . It is necessary first to isolate the bis-
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
muth in a concentrated form, since the most sensitive bis- 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 ap-
parent 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 concentra-
tion between 0.2 and 3.0 mg. of bismuth per 100 cc. may be em-
ployed.
Two cubic centimeters of the properly adjusted bismuth solu-
tion 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 (MgCla-
6H20), and 37.5 grams of sodium dihydrogen phosphate (NaH2-
POe,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 bis-
muth standards used to derive the calibration curves. This solu-

1,,,11 [ , ( I 1 , , ( , 1
0.02 005 010 015 0.20 PER lo0 CC.
IxlcT4 .?*IOq4 3#d4 +&ON 7HE ARC
BISMUTH CONCENTRATION (Me ) 1.40 - I L
I ”
FIGURE
1 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 dis-
advantage 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 bis-
TABLEI. BISMUTH
RECOVERIES muth concentration. The alternative, when dealing with
Bismuth added, quantities above 0.20 mg. of bismuth per 100 cc., is to deter-
Bismuth
mg. found, 0 . 2 5 0.50 ‘.O0 2.00 mine the average density of the bismuth line corrected for
mg. :
O.Ool O.O1
:::
: 5::: : : : : 8 ii 8 i: !; :: variations in the density of the internal standard and to evalu-
ate 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
intravenously)
Tissue Weight Bismuth Found
Gams Mo.
Blood 76.1 0.001
Applicability, Sensitivity, and Accuracy of
Liver 54.8 0.013 Method
Spleen 0.7 0.000
Kidneys 11.5 0.08
The recoveries for known amounts of bismuth added to
Heart 4.1 0.000
Lungs
Central nervous system 6.2
14.0 0.001
0.000
digested portions of beef are tabulated in Table I. In Table
Testes 1.4 0.000 I1 are recorded the results obtained from the tissues and
Bile ... o.ooo ’ excreta of an experimental animal. The weights of tissue
Pancreas 2.1 0.004
Muscle
Bone 578.5
132.0 0.014
0.18 available have been recorded in order to indicate the size of
Skin 208.4 0.01 samples which can be utilized. The error of analysis, as
Intestinal tract 132.2 0.05
shown in Table I, naturally depends on the quantity of bis-
Contents of tract 164.0 0.03
Ears, tail, and feet
Remainder 130.0
348.0 0.10
0.05 muth encountered. For amounts above 0.20 mg. the error
Entire carcass
~

0.633
Urine
Feces 2.24
0.90
Total bismuth found in excreta - 3.140
__
Total bismuth found 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 in-
candescent 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 de-
scribed for lead (I), while t,he region A2600 to A3800 is
photographed.
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
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 suf-
ficiently 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
(1) Cholak, J., IND,
E ~ GCHEM,,
, Anal, Ed,, 7, 287 (1935).
( 2 ) Cholak,
J., J . A ~Chem., sot., 57, 104 (1935).
(3) Lundegardh, H., “Die quantitat>ive Spektralanalyse der Ele-
mente,” P a r t 11, Jena, G u s t a v Fischer, 1934.
(4) Olseh, J. c.9“Chemical Annual,” 7th ed., P. 477, NOW York,
D. Van Nostrand Co., 1934.
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 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 pos-
sible loss while decanting and from creeping of the pre-
cipitate.
By the use Of 30-cc*sintered-glass Buchner
nation 3 G. 4),this centrifuging and decanting can be avoided.
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 Present addresa, 44 Irving St., Cambridge, Mass.
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.
Very satisfactory results were obtained with this method
which lends itself well to fast routine work,
Literature Cited
(1) Rymer, M.R., and Lewis, R. C., J . Bid. Chem., 95, 441 (1932).
(2) Tisdall, F. F., Ibid.2 56, 439 (1923).
RECEIVED
September 1, 1936.