Application of a Photoelectric Colorimeter Determination of Bismuth in Biological Materials RE.IVIS C. SPROULL AND ALEXANDER 0. GETTLER, New York University, New York, N. Y.
T
Mfiller (5) has shown that the basis for every colorimetric method should be the Lambert-Beer law. In order to establish the justification for the use of this system in a colorimetric procedure the following solutions were prepared, and experiments performed. All reagents were prepared from chemicals of A. C. S. . . specification. STANDARD BISMUTH, 0.2321 gram of bismuth nitrate, Bi(KO&.5H20, dissolved in 50 ml. of 1 to 10 nitric acid and diluted to 1 liter to give a solution containing 10.00 mg. of bismuth per 100 ml.
HE potassium iodide method for the determination of
bismuth in biological materials has been used in many forms since first applied by Autenrieth and Meyer (I) and modified by Leonard (4). It depends upon the formation of a yellon- bismuth iodide complex in acid solution and in the presence of a reducing agent. Scholtz and Chaney ( Y ) , without evaluation of the effect of the reagents on the optical properties of the solution, applied a photoelectric comparator to this method, using standard bismuth solutions for calibration. Baggesgaard-Rasmussen, Jackerott, and Schou (2) showed by means of a Koenig-Martens spectrophotometer that the complgs involved absorbs blue light u p to a wave length of 4500 .1. They found, furthermore, that variation of the acid and potassium iodide within narrow limits had apparently no effect on the light absorption. I n this investigation an attempt has been made to evaluate objectively, by means of a photoelectric colorimeter, the principal factors which affect the application of the iodide method. Revised procedures are proposed for the determination of bismuth in urine, kidney, liver, and muscle, and test analyses are included to indicate the accuracy.
1
z
1
1.990
0 1.980
9
f
16 c
All determinations were made with a photoelectric photometer employing a single cesium cell and standard substitution technique. Bpproximately monochromatic light was supplied hy means of a 6-volt incandescent bulb operating on a storage battery (Exide, Type LXGH), a blue Pyrex glass filter (Corning S o . 554 H. R. Lantern Blue), 6.0 mm. in thickness, and a heat filter (Corning No. 396, Light Shade Aklo). The blue filter was chosen because its maximum transmission (3) occurs in the region of the prominent absor tion band of the bismuth iodide complex (a). Other blue fiiers gave less satisfactory results. Absorption cells of 1.0-cm. or 2.0-cm. depth with optically flat windows were used to hold the solution and the water, respectively. Measurements of the photocurrent were made with a vacuum tube voltmeter working on a 6C6, R. C. A. tube, with a 6E5 tube as null point indicator. This applies the principle of the 6F5 circuit of Mriller (6). The instrument used in this work gave a precision of *0.2 millivolt, equal to 1 0 . 1 per cent of the color measured.
1.910
1960
L9M)0
I
2 3 4
3 6
7
8
9
IO II
I2 I314
GAMMA B I S M U T H P E R IO
ML.
FIGURE2. RELATIONSHIP BETWEEN LOGARITHM OF PERCEKTTRASSMISSIOX AND 12 MICROGRAMS OR LESSOF BISMUTH Concentrations of reagents were same as in Figure 1. Cells of 2.0-om. thickness were substituted for those of 1.0-om. thickness. Symbols represent determinatlons made a t different times on known concentrations of bismuth. Differences observed a t a fixed concentration are a measure of precision of method.
SODIUM SULFITE,1.00 ram dissolved in water, acidified with 0.8 ml. of concentrated sufffuric acid, and diluted to 200 ml. This
solution must be freshly prepared every day. POTASSIUM IODIDE, 17.0 grams dissolved in 1 liter of water. ACID, 280 ml. of concentrated sulfuric acid diluted SULFURIC to 1 liter. Varying amounts of the standard bismuth solution were mixed in each case with 50.0 ml. of sulfuric acid solution, 13.4 ml. of sodium sulfite solution, and 16.8 ml. of potassium iodide solution, and the whole was diluted to 100 ml. in stoppered volumetric flasks. These amounts of reagents were used to get a wide range of bismuth concentration within a measurable transmission. The percentage transmission was then determined for each solution.
FIGURE
Figures 1 and 2 show that the relationship between the logarithm of the percentage transmission and the bismuth concentration is linear; thus, the Lambert-Beer law is obeyed for concentrations u p to 2.00 mg. of bismuth per 100 ml. of solution. Figure 2 was prepared as a basis for the determination of from 1 to 10 micrograms of bismuth in 10 ml. of normal blood serum. Absorption cells of 2-cm. thickness and 8-ml.
RELaTIONSHIP BETWEEK LOGARITHM O F P E R CENT TR.4h'sMISSIOK AKD BISMUTHCOSCENTRATIOS
1.
For each 100 ml. of solution, 50.0 nil. of 28 per cent ( b y volume) sulfuric acid, 13.4 ml. of 0.5 per cent XatSOa solution, and 16.8 ml. of 1.7 per cent I i I solution were used with varying amounts of standard bismuth nitrate solution.
462
July 15, 1941
ANALYTICAL EDITION
463
amounts of iron as feiric sulfate were u-ed; the quantities of reagents added were the Same as those used in obtaining the transmission curve (Figure 1). Curves IV and ITA, Figure 4, show that the concentration of iron may be as high as 20 mg. nitliout interfering with the determination. T n enty milligrams or less of iron do not cause a change on standing several hours, whereas more than 20 mg. cause the precipitation of iodine. Separate test analyses with knov-n concentrations of bismuth and various quantities of heavy metals showed that quantitative recovery of bizmuth may be obtained in the presence of the following concentration per 100 ml. of heavy metalp: 2 ing. of lead, 2 mg. of mercury, 0.5 mg. of copper, and 10 mg. or more of arsenic. As little as 0.5 mg of silver interferes. Thus it was concluded that the concentration of the above heavy metals normally appearing in tissue will not interfere unless silver is present. Test analyses showed that, if the solutions are kept securely stoppered after mising the reagents n i t h the bismuth, the yellow color is stable for as long as 14 days. The same percentage transmission vas found a t the end of this time a i imniediately after preparing the color.
-1pplication of the Method to -1iial>ses of Biological -Material 1
I
4
8
I
IL
1
16
1
?.O
1
24
1
28
I
32
I
36
+!
ML. OF REAGENT PER 100 ML. OF SOLUTION FIGURE
3. TNFLCESCE
O F VARIATIONS O F REaGENTS RECOVERY OF BISMUTH
UPOX
One reagent was Yaried in each curve while others were in same concentrations as in Figure 1 . I , 1.1. Influence of concentrated sulfuric acid upon recovery of 0.400 and 0.800 nig. of bismuth per 100 ml., respectively 11. 11.4. Influence of 0.5 per cent sodium sulfite 111. IIIA. Influence of 1.7 per cent potassium iodide
volume were used. T o reduce the accidental errors several measurements were made for each concentration on this curve. The lines do not intercept the axis of ordinates a t 100 per cent transmission, because distilled water instead of the reagents (which absorb some light) was used in the reference cell, in order to avoid making u p a synthetic mixture of reagents for each determination. Furthermore, if such a mixture were allowed to stand in the open cell i t would slowly lose sulfite and free iodine might be liberated. Thus water furnishes a perfectly reproducible reference cell, but by itq use 110 error is introduced into the determination. To determine the relative effects of the reagents on the color, fixed quantities of standard bismuth solutions were taken, and the concentrations of one of the reagents were varied while all pther concentrations were kept the same as in the preparation of the Lambert-Beer's law curve. Two bismuth concentrations were used for each study0.400 and 0.800 mg. per 100 m1.-and the results are shown in Figure 3. Curves I11 and IITA indicate that the color intensity is a function of the iodide concentration, but as shown by cuives I, IA, 11, and IIA, it is affected only slightly bv the acid and the sulfite. Some sulfite must be Dresent to prevent the precipitation of free iodine, as evidenced by the sudden initial fall 11 and IIA) in the sulfite If the iodide is in great excess over the sulfite free iodine is liberated, resulting in light absorption. The arrons in Figure 3 indicate the concentrations of &e reagents used in obtaining the Lambert-Beer's curve (Figure 1). To det'ermine the concentration of iron which will interfere, 0.400-mg. and 0.784-mg. samples of bismuth and variable
URITE. To 100 ml. of urine in a Kjeldahl flask, 10 ml. of concentrated nitric acid and 7 . 5 ml. of concent'rated sulfuric acid are added. This is evaporated to fumes of sulfur trioxide over an open burner. While fuming, a mixture of nitric and perchloric acids (1 to 2) is added dropwise until all color is destrored. The excess nitric acid is now destroyed by the addition of 1 ml. of 30 per cent hydrogen peroxide, dropwise, to the hot mixture. The peroxide is in turn removed by vigorous boiling. The flask is allowed to cool, about 10 ml. of water are added, and the solution is again evaporated to fumes of sulfur trioxide. The contents of the flask are now rinsed into a 50-nil. volumetric flask, usin not more than a total of 25 ml. of water. After cooling, 6.7 m?. of 0.5 per cent sodium sulfite and 8.4 ml. of 1.7 per cent potassium iodide are added and diluted to 50 ml. The solution is centrifuged if not perfectly clear and is then ready for the photometer. The potentiometer is set at zero mid the instrument, is adjusted to a null point with the phototuhe dark. v
0
'0
m 09
z? 08 n w cc
07
52
04
w06
05
I O 3
5 02 2 Eo1 0
E
4
8
12
M G OF Fe*"
b
20
24
26
2?
35
40
PER 100 ML OF SOLdT O h
FIGURE 4. ISFLCESCE O F I R O N , hS F E R R I C SULFATE, UPON RECOVERY O F 0.400 A S D 11.754 MG. OF BISVUTHPER 100 1 1 ~ . The t n o absorption cells are filled with ~ ~ i t ethe r , constant light source is turned on, and the potentionletel i q set to a null point. Readings for the right cell, Ro,and the lett cell, LO,are taken them back and forth eo as t o by cept the beam of light. Three such readinas for each cell are, in-general, satisfact&y. If the readings f& the two cells are not the same, a correction is applied in the calculations. The left cell is emptied, rinsed n.ith the solution, and then filled with the solution. ~~~i~ alternate readings for the unknotvn,R, and for the water, RO,are taken.
Vol. 13, No. 7
INDUSTRIAL AND ENGINEERING CHEMISTRY
464
TABLE I. DETERMINATION OF BISMUTH Volume of Urine
MI. 100 50 50 100 100 50 100 100 50 100 50 100 50
100 50 50
Bismuth Added Mg.
Bismuth Found
Recovery
Volume of Urine
Mg.
7%
M1.
Recovery from Urine 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0,003 0.051 0.050 0.098 0.100 0.105 0.100 0.148 0.150 0,202 0.200 0,251 0.250 0.301 0.300 0.348 0.350 0.397 0.400 0.445 0.450 0.493 0.500 0.599 0.600
Bismuth Added M9
Muscle, Cont'd 12
102:o 98 0 105 0 98 7 101 0 100 4 100 3 99 4 99 3 98.9 98.6 99 8
Av. 100.1 Mean deviation, % ' 1.3
13 13 22 13 19 20 15 19 22 17 19 20 24 12
Bismuth Found
M g.
.
%
Recovery from Tissues, Cont'd
0,050 0.100 0.150 0.200 0.300 0.400 0.400
0.048 0.099 0.153 0.200 0.304 0.398 0.405
0,500
0.500
0,900 1,000
0.910 0.973
96 0 99.0 102.0 100.0 101.3 99.5 101.3 100.0
101.1 97.3 Av. 100.8 Mean deviation, % * 1.5
Recovery from Tissues Kidney
Recovery
Recovery from Blood Serum
Grams 22 8 19 10 9 8 9 21 23
0.000 0.000 0.100 0,200 0,200 0.300 0.400 0.600 0,800
0,000 0.000 0.102 0.204 0.196 0.315 0.393 0.630 0.806
Liver 20 23 25 27 25
0.000
0.000 0.000 0.200 0.300
0.000 0.007 0.000 0,208 0.306
104:o 102.0
Muscle 13 17
0.000 0.000
0,002 0.001
... ...
Blood Serum
...
102.0 102.0 98.0 105.0 98.3 105.0 100.8
M1.
Micrograms
10
0.0 0.0 0.0
0.0 0.0 0.0 2.0 2.0 4.0 4.0 6.0 6 0 8.0 8.0 10.0
... ...
Micrograms 0.0 0.3 0.0 0.4 0.0 0.0 1.8 1.9 4.0 4.3 6.2 5.0 8.1 8.6 9.6
90 95 100 108 103 83 101 108 96 10.0 100 Av. 98 Mean deviation, % *6
10.0
The per cent transmission of the sample is calculated as follows: Cell correction = Ro!Lo, er cent transmission = &/Lo x RIR, , - .X. 100. where this Rnhn is usuallv eaual to 1. The bismuth concentration in mg. per"l0dml. is found by interpolation Of the Beer's law curve (Figure The bismuth content of the sample analyzed is given by: Mg. of Bi
...
... ... ... ... ..
lowed. Less than 1 microgram of bismuth should not be estimated by the method. Known amounts of bismuth were added to a series of tis(cat and rabbit), urine (human), and blood serum (dog) samples. Control experiments using normal tissue, to which
b 100
= a X -
where a = mg. of bismuth in 100 ml. obtained from the Beer's law curve (Fi ure 1) and b = total volume to which digested material was %luted. LIVER, KIDNEY, AND MUSCLE. Not more than 25-gram samples are placed in an Erlenmeyer flask and covered with a volume of nitric acid equal to about twice the weight of the tissue. The flask is heated gently on a steam bath until a clear brown or yellow solution results. On cooling, a layer of fat will usually separate, and is removed by filtering throu h glass wool into a 500-ml. Kjeldahl flask. The flask and the $ass wool are rinsed with nitric acid and the rinsin s added to the filtrate. A volume of 7.5 ml. of concentrated s$furic acid is added to the Kjeldahl flask and the mixture evaporated to fumes of sulfur trioxide. The digestion and estimation are now finished as in the procedure for urine. BLOODSERUM. To 10 ml. of normal serum in a 100-ml. Kjeldah1 flask, 1.5 ml. of concentrated sulfuric acid and 5 ml. of concentrated nitric acid are added. This is digested as described for urine. By means of 5 ml. of water the residue is rinsed into a 10-ml. volumetric flask and cooled, 1.4 ml. of 0.5 per cent sulfite and 1.7 ml. of 1.7 per cent otassium iodide are added, and the solution is diluted to 10 mf This is centrifuged and the percentage transmission determined as for urine, substituting cells of 2.0-cm. depth for those of 1.0-cm. depth. The bismuth concentration in micrograms per 10 ml. is found by interpolation of the Beer's law curve (Figure 2). For the determination of such small quantities of bismuth all lassware used should be first washed with hot nitric acid, all iasks should be kept covered to protect against dust particles, and the rigorous precautions for exact photometry must be fol-
TABLE 11. DETERMINATION OF BISMUTH Total Yolume
Bismuth Added
MI.
Mg.
908
2.50
Aliquot Recovery from Urine
50 50
Total Bismuth Found Mg.
Recovery
E$ Av. Mean deviation, %
100
Unknoan
200
Unknown
10 10 10 10
Total Weight
Recovery from Tissue (Liver)
Grams 242
Grama 25
262
29 1
3.10 3.08 5.08
70
95.2 94 8 99 3 -2
..
...
...
5.20
...
2.57 2.59 1.98 1.98 2.58 2.51
...
... ... ...
...
...
ANALYTICAL EDITION
July 15, 1941
no bismuth was added, were also conducted. The analytical results are given in Table I. The complete procedure was put into the hands of a technician who obtained the results of Table I1 in duplicate and triplicate on known and unknown samples. These figures are submitted as evidence that the method is adaptable to clinical use in the hands of a technician. Results on other tissues have been found as precise as these shown.
Summary By means of a photoelectric colorimeter, the principal factors which affect the application of the potassium iodide method for the determination of bismuth in biological materials were evaluated. Procedures for a photoelectric determination of bismuth in urine, kidney, liver, muscle, and blood serum have been
465
devised with test analyses included to indicate the accuracy of such procedures.
Literature Cited (1) Autenrieth, W., and Meyer, A., MQnch. med. Wochschr., 71, 601 (1924). (2) Baggesgaard-Rasmussen, H., Jackerott, K. A . , and Schou, S. A., Biochem. Z., 193, 53 (1928); Dansk. Tids. Farm., 1, 391 (1927). (3) Hodgman, C. D., “Handbook of Chemistry and Physics”, 19th ed., p. 1563, Cleveland, Ohio, Chemical Rubber Publishing Co., 1939. (4) Leonard, C. S., J . Pharmacol., 28, 81 (1926). ( 5 ) Muller. R. H.. IND.ENG.CHEM..Anal. Ed.. 7.. 223 (1935). . . (65 Ibid., 11, 9 (1939). (7) Scholtz, J. R., and Chaney, A. L., Am. J. Syphilis Gonorrhea Venereal Diseases, 23, 759 (1939). SUBMITTED in partial fulfillment of the requirements for the degree of doctor of philosophy a t New York University.
Perchloric Acid Oxidation of Organic Phosphorus in Lake Waters REX J. ROBINSON Oceanographic and Chemical Laboratories, University of Washington, Seattle, Wash.
T
H E utilization of phosphate, dissolved in lake water, by growing phytoplankton attaches considerable importance to this element and to the phosphorus in the lake. Various studies have shown that only a small portion of the total phosphorus content is present as the readily available phosphate but a large potential supply exists organically bound. The Deni 6s ( 1 ) colorimetric method estimates only the phosphate phospforus. The amount of organic hosphorus is derived by deducting the phosphate hosphorus Prom the total phosphorus content. The methof as described by Robinson and Kemmerer (8) has served very well for the determination of total phosphorus. Briefly, the organic material in a 100-ml. water sample is digested with 0.2 ml. of concentrated sulfuric acid and 0.5 ml. of concentrated nitric acid t o convert the organic phosphorus to phosphate. Shortly before sulfuric acid fumes appear 3 ml. of concentrated hydrochloric acid are added t o destroy the excess nitric acid. Although the above method has proved its value, the following alternate procedure is proposed because of its greater simplicity. To the 100-ml. water sample contained in a 125-m1. Erlenmeyer flask, 0.2 ml. of 72 per cent perchloric acid is added and the sample is evaporated to fumes of perchloric acid. (More acid must be added to hard waters to gain an excess of the acid.) If oxidation of the organic material is slow, as is sometimes the case with water from extractive lakes, the flask may be covered with a watch glass t o prevent loss of perchloric acid and the heating continued until the coloration disappears. The use of perchloric acid as the oxidizing agent has a number of advantages. Only one acid, rather than three, is measured. Undesirable fumes are not inhaled during pipetting or discharged during digesting. The excess nitric acid must be completely removed, as it causes fading of the blue phosphomolybdate complex; the excess perchloric acid does not cause fading. The flask may be covered with a watch glass during digestion to prevent loss of perchloric and phosphoric acids; this cannot be done with the nitric acid because
of the retention of the nitric acid. Consequently, with a covered flask there is less need for careful temperature regulation in the digestion to prevent loss of phosphoric acid. The nitric acid is destroyed and expelled a t a low temperature, the perchloric acid is present throughout; this means that difficultly oxidizable substances may be subjected to a higher oxidation temperature. Lastly, there is less phosphate impurity in perchloric acid than in the sulfuric-nitric-hydrochloric acid mixture. The perchloric acid oxidation has proved an efficient method of oxidizing organic phosphorus compounds. First it has been checked by the analysis of Eastman’s c. P. glycerophosphate, the phosphorus content of which had been verified by oxidation and gravimetric analysis of macrosamples. Amounts of glycerophosphate approximating the phosphorus content normally found in 100 ml. of lake water were used to simulate lake water conditions. Complete recovery was obtained, as indicated in Table I. Secondly, the efficiency of the method has been checked by analysis of lake waters which were also analyzed by the sulfuric-nitric-hydrochloric acid method. A comparison of the two methods is included in Table 11. Trout Lake water was particularly suited to a critical comparison of the two methods, because in it there was enough extractive organic material to make it more difficult to oxidize than the average lake water. The organic material was as quickly and efficiently oxidized by the perchloric acid as b y the nitric-sulfuric acid mixture. During the progress of this
TABLE I. ANALYSISOF GLYCEROPHOSPHATE Phosphorus Taken
Phosphorus Found
Mg./l.
Mg./1.
0.028 0.02s 0.028 0.028
0.029 0.027 0.027 0,027
