Microdetermination of Arsenic in Biological Material JAMES A. SULTZABERGER, Research and Biological Laboratories, Parke, Davis & Co., Detroit, JIich.
,
I
hydroxide, dilute, and adjust with N hydrochloric acid to pH 6.5 to 6.8. Add sodium bicarbonate to about pH 7.2 and dilute to 1 liter. This solution, containing 1 mg. of arsenic per ml. as sodium arsenite is stable in that form for many months. Ten milliliters o i this stock solution diluted to 1 liter contain 10 micrograms per ml. COLOR-DEVELOPING SOLUTION.Solution I. Dissolve 1 gram of ammonium molybdate in about 45 ml. of distilled water, add 50 ml. of approximately 10 N sulfuric acid, and dilute to 100 ml. This solution is stable for several weeks. Larger crystals of ammonium molybdate appear to yield a lower blank. Solution 11. Take 20 ml. of Solution I, dilute to about 90 ml., and shake. Add 2 ml. of 0.15 per cent hydrazine sulfate solution, dilute to 100 ml., and mix well. This solution is freshly prepared daily.
N THIS laboratory, many satisfactory arsenic determinations have been made by distilling arsenic tri-
chloride and titrating with iodine according to Bang ( I ) .
Since the end point of this method is not sufficiently sensitive for minute quantities of arsenic, the method was modified by trapping the arsenic trichloride in dilute nitric acid and determining as molybdenum blue according to Sobotka (6),whose method was based on that of Morris and Calvery ( 3 ) . Rodden (4) determined arsenic as the trichloride in ferrous and nonferrous alloys by distilling it in a stream of carbon dioxide into water, oxidizing with nitric acid, evaporating and drying a t 130" C., and reading the molybdenum blue color photometrically. Hubbnrd (f?) adapted the method to
Procedure PREPARATION OF THE SAMPLE.The sample or an aliquot is di-
biological material.
gested with sufficient nitric acid and 5 ml. of sulfuric acid as in the procedure of Bang ( 1 ) or Morris and Calvery ( 3 ) . Care is taken to maintain an excess of nitric acid until all halogens are removed and all organic matter is oxidized. About 20 ml. of distilled water are added to the cooled digest, which is heated until the sulfuric acid fumes strongly, in order to remove any remaining oxides of nitrogen. In the decomposition of bone, the oxidation is facilitated by dissolving the sample in reagent fuming nitric acid, specific gravity 1.5, diluting with an equal volume of water, and extracting the fat twice with ether. The residue of the combined extracts is digested separately as above and added to the original nitric acid solution. The calcium sulfate is filtered off and washed well and the digestion of the filtrate is completed as usual. Any loss of the 5 ml. of sulfuric acid is replaced. DISTILLATION.The Fresenius flask (Figure l), employed as the receiver and containing 1 ml. of concentrated nitric acid and about 8 ml. of distilled water, is surrounded by an ice-water bath and attached to the connecting tube for distillation. (The amount of distilled water required depends on the construction of the Fresenius flask. It should be sufficient to trap the arsenic trichloride and yet not fill more than two thirds of the lower bulb before bubbles of gas are released when the nitric acid solution is under gradual pressure from the distillation.) The following salts are previously placed in a small covered beaker: 4 grams of potassium chloride, about 0.4 to 0.5 ram of potassium bromide, and 1 gram of ferrous ammonium sulkte. The digest, in 5 ml. of concentrated sulfuric acid in the Kjeldahl flask, is diluted with 6 ml. of distilled water and chilled under the tap. The salts are added all a t once through a short wide-stemmed funnel to the flask which is connected immediately for distillation. A strong flame is applied directly to the flask until boiling starts, when it is reduced a t once until the tip of the flame just touches the flask. When the effervescence begins to subside and the salts are about half dissolved, the flame is gradually increased until the salts are all dissolved and the solution is boiling rapidly. Meanwhile, the solution in the Fresenius flask is kept under strict observation. When the first drop of liquid has fallen from the connecting tube into the receiver, the distillation is stopped. The whole time required for this distillation should not be more than 2 to 3 minutes. The receiver is removed and the nitric acid solution is transferred to a 25-ml. Erlenmeyer flask by pouring the solution through the bulbs of the Fresenius flask. The flask is washed twice with 1ml. of distilled water which is added t o the distillate. An aliquot may be taken at this point, especially if the arsenic content is entirely unknown. The nitric acid solution is transferred from the receiver to a 25-m1. glass-stop ered volumetric flask and diluted to the mark, and an aliquot or10 ml. or less is taken for evaporation, If it becomes necessary to take another aliquot for evaporation, this should be done within 24 hours, because this solution is somewhat unstable after that time. Otherwise, the aliquot should be evaporated with sulfuric acid and redistilled. The size of the aliquot should be such that it contains less than 100 micrograms of arsenic. Spinal fluid and blood containing very small amounts of arsenic should be analyzed directly without taking an aliquot. In this case it is desirable to
m 1.
FIGURE 1. FRESEXIUS FLASK
In the method described below, arsenic trichloride is distilled rapidly in a stream of hydrogen chloride and trapped in dilute nitric acid. Apparatus Kjeldahl flasks, 300 ml., and a Fresenius flask (Figure l), 25 ml. Bent connecting tube with rubber stoppers; inside diameter of tube, 4.5 mm. The middle section of the tube slopes slightly toward the receiver. Erlenmeyer flasks, 25 ml., glass bulbs, electric hot plate, thermostaticallv controlled (Fisher Scientific Co., AutemD heater). and an electric oven. ' Cenco Photelometer (photoelectric colorimeter) with a 625millimicron (red) filter and 1-cm. cells with a volume of about 8 ml. . I
Chemicals Sulfuric acid, specific gravity 1.84, arsenic-free; nitric acid, specific gravity 1.42, arsenic-free. Potassium chloride, potassium bromide, ferrous ammonium sulfate (Mohr's salt), ammonium molybdate, and hydrazine sulfate (Merck's reagent).
Reagent Solutions STAXDARD ARSENICSOLUTIONS.Dissolve 1.32 grams of arsenic trioxide (Bureau of Standards) in 30 ml. of N sodium
408
ANALYTICAL EDITION
June 15, 1943
409
obtain i t sufficiently pure, the blank should be determined until experience has shown that i t is unnecessary.
Calibration Curve It was found more practical to plot the calibration curve as shown in Figure 2 on the basis of the arsenic concentration
20
40
60
versus the photoelectric colorimeter readings than against color density values which must be calculated from instrument readings. I n practice, two curves are employed, one from 0 to 15 micrograms and the other from 15 to 100 micrograms. The light filter first employed in the photoelectric colorimeter in this method to measure the absorption of arsenic color reaction had a maximum transmission band at about 625 millimicrons. The bands employed in other methods have been in the range of 567 to 725 millimicrons. Very recently the maximum absorption of the arsenic color reaction was determined as shown by the curve in Figure 3. It is obvious that the maximum absorption is in the infrared at about 840 millimicrons. When a dark infrared gelatin filter was employed in the photoelectric colorimeter, the sensitivity mas doubled. A glass filter having the proper wave band is now being obtained. Jl'hen it arrives, it will be standardized. If i t has the maximum transmission a t the proper wave band, a new calibration curve will be prepared.
a0
MICROGRAMS ARSENIC
FIGURE 2. CALIBRATION CURVE use .sufficiently pure distilled water to reduce the blank to a minimum. EVAPORATIOS ASD DRYISG.The nitric acid solution in the open Erlenmeyer flask is evaporated almost to dryness as soon as possible and not alloFed to stand overnight, because it is easily contaminated with laboratory vapors. This evaporation is done on a thermostatically controlled hot plate. A temperature of 120 to 130" C. is maintained as read on a thermometer suspended in oil in a 30-ml. beaker in the center of the hot plate, so that the bulb of the thermometer touches the bottom of the beaker. (When the evaporating solution has mostly disappeared the temperature may rise suddenly above 130" C. If the temperature is adjusted to 120" to 125" C., it is not apt to rise above 130" a t the end of evaporation.) Drying is completed by placing the Erlenmeyer flask in an electric oven at 120" C. for 1 hour. This residue is very stable. DEvELoPmvT OF COLOR. Exactly 10 ml. of the color-developing reagent solution I1 are added to the dry residue in the 25ml. Erlenmeyer flask, which is covered with a glass bulb and heated in a water bath for 10 minutes a t 80" to 90' C. After cooling under the tap, the colored solution is transferred to a 1-cm. cell and compared with water in the comparison cell a t a reading of 100 in the photoelectric colorimeter with a 625-millimicron (red) filter. To the reading thus obtained is added a reagent blank correction, which is determined with each set of analyses. From a curve, previously prepared by plotting the corrected Photelometer readings from the distillation of known amounts of arsenic, the amount of arsenic in the sample is determined. THEREAGEXT BLAXK.The amount of the r blank to be added is determined by placing 5 ml. of concentrated sulfuric acid and 6 ml. of distilled water in a Kjeldahl flask, distilling, evaporating, and reading as in other determinations. The reading from the blank is % subtracted from 100 to obtain the blank correction which is added to the readings from known or unknown amounts of arsenic.
Test of the Method
O
a separate reagent bottle, where i t is protected from fumes evolved from digestion processes. Distilled water employed in the receiver and in preparing the color developing solutions should be pure and protected from laboratory fumes. Although these fumes do not usually contain arsenic, they increase the color in the blank. When
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Arsenic was added to 10-ml. samples of whole blood (Table I) to 50-ml. samples of normal urine (Table 11) and to normal mice (Table 111). The recovery of arsenic in the three tables is 100 * 5 per cent except with 1 microgram, where the limits are * 10 per cent. Hence, the accuracy of the method is about 0.2 microgram in the lowest range. KO arsenic was found in samples of blood to which none was added. As arsenic is usually found in normal urine, i t appears in Table I1 as a sample blank, although the reagent blank has been reduced to a minimum. I n Table 111, a sample blank is also found. I n order to identify this as arsenic and not phosphorus, which also gives the molybdenum blue color, these tests were repeated and a double distillation was performed in order to remove any
Absorption Curve Reacfion
Arsenl'c Co/or
Beckman Specftvphofome f e r
i \
0.4 0.2 -
I
400
1
500
600
700
800
QOO
Wave Lenqth in Millimicrons
times necessary to redistill the mater to
FIGCRE 3. ABSORPTIONCURVE
1000
INDUSTRIAL AND ENGINEERING CHEMISTRY
410
'FABLE
1. RECOVERY O F ARSENICI N
\THOLE
BLOOD
T.4BLE
11. RECOVERY O F ARSENIC FROM
(lo-ml. samples)
KO. 1
2 3 .4 5 6
7 8
9 10 11 12
Arsenic Added
Reagent Blank
Arsenic Found
Micrograms
Microgram
Micrograms
0.0 0.0 0.0
0.2 0.2 0.2 0.2 0.3 0.2 0.2 0.3 0.3 0.3 0.3
1 .o 1 .o
5.0 10.0 15.0 30.0 40.0 60.0
80.0
0.3
0.2 0.2 0.2 1.2 1.2 5 3 10.0 15.4 28.8 39 8 63.3 78.8
(50-1111. Arsenic Recovered
Micrograms 0.0 0.0 0.0 1.0 0.9 5.1 9.8 15.1 28.5 39 5
63.0 78.5
1oo:o 90 0 102.0 98.0 100.7 95.0 99.0 105.0 98 1
phosphorus wliich might be present in the first distillate. Those mice to which no arsenic was added still gave SUI:stantially the same results. Hence, phosphorus was completely eliminated in the first distillation, although it was accumulated from the bones in a relatively large quantity.
Discussion The apparatus employed in this method, except the photoelectric colorimeter, is of the simplest type. K i t h a good hood there is no necessity for ground-glass joints or other expensive equipment. Since the actual distillation requires only 2 to 3 minutes, only one set of distillation apparatus is required. This consists of nothing more than the Kjeldahl flask employed in the digestion, a bent connecting tube with rubber stoppers a t each end, and the small Fresenius flask used as the trap. The time of evaporation of the distillate has been decreased by employing a thermostatically controlled hot plate instead of a steam bath. At 120" to 130" C. there is no loss of arsenic during evaporation. If the temperature of the hot plate is allowed to rise to 130" to 135" C., there is very little if any loss and the time of evaporation is about 1.5 hours. At 140" C. or above the loss of arsenic becomes appreciable. Because of the total savings in time, six to eight samples of blood or urine may be analyzed in an 8-hour day. KO interference was caused by the presence of antimony, bismuth, selenium, or phosphates either in the blank or in the recovery of arsenic. The amount of arsenic in the reagents employed appears to be negligible. I n the separation of arsenic by the generation of arsine as in the Gutzeit or Marsh tests, the use of zinc or tin is required. These metals are difficult to obtain entirely arsenic-free in normal times and now the exigency is greatly increased. If the dilute nitric acid employed in the trap and the water employed in the color-developing solution are sufficiently pure and protected from laboratory fumes, the blank may be reduced to zero. No filtration nor adjustment of the p H value of the colordeveloping solution is necessary. This solution is placed directly in the dried Erlenmeyer flasks. The characteristic molybdenum blue color produced by heating is stable and obeys Beer's law, as shown when the color density is plotted against the arsenic concentration. The readings in this procedure are made on the transmission through 1-em. cells. The sensitivity may be increased by placing the ssme volume of the arsenic solution in longer cells. It is expected also that the sensitivity will be more than doubly increased by the employment of the new absorption filter which is being obtained. Although the range of the color test will be decreased, that is not serious, since a smaller aliquot can be taken. It is planned to continue the investigation of these items and to report in a subsequent paper. Although this method has been employed successfully in the analysis of about a thousand samples of various
1 2 3
Reagent Blank
Micrograms 0.0
Microgram
Micrograms
70
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1
1.6 1.4 1.4 2.4 2.6 2.6 6.7
...
0.0 0.0 1.0 1.0 1.0 5.0 5.0 10.0 15.0 20.0
4 5 6
7 8 9 10
11 12 13 14
40.0
60.0 80.0 Reagent blank is included.
TABLE
No. 1 2 3 4 5
6
CRINE
samples)
.4rsenic Added
KO.
5%
Vol. 15, No. 6
Arsenio Recovered
... ...
90.0 110.0 110.0 104.0 102.0 97.0 101.3 100.0 105.0 102.5 98.9
6.6 11.2 16.80 21.5 43.6a 63.1a 80.5
0.1 0.0
111. RECOVERY O F ARSEXIC
FROM S O R X 4 L
hfICE
Weight
Arsenic Added
Reagent Blank
Arsenic Found
Grams
Micrograms
Microgram
Micrograms
Micrograms
19 20 18
0.0 0.0 10.0 20.0 30.0 40.0
1.4 1.4 11.0 20.9 29.9 40.7
1.0 1.0 10.6 20.5 29.5 40.3
17
18 18
0.4 0.4 0.4 0.4 0.4 0.4
Arsenic Recovered I
.
96:O 97.5 95.0 98.2
hiological materials, it is also applicable to any other type of analysis where the microdetermination of arsenic is required.
Conclusion A simple, rapid, and accurate method has been developed for the microdetermination of arsenic in biological materials, including blood, urine, bone, and tissue. The sample is digested with concentrated nitric and sulfuric acids, and the arsenic is distilled as the trichloride and trapped in dilute nitric acid solution in a Fresenius flask. The characteristic molybdenum blue color is developed from the evaporated residue and read in a photoelectric colorimeter with a 625millimicron filter. The range is 0 to 100 micrograms of arsenic. Based on the recovery of arsenic, the accuracy is about 0.2 microgram in the lowest range. Very recently the maximum absorption of the arsenic color reaction has been found to be in the infrared a t about 840 millimicrons. It is expected that the employment of this wave-length band will more than double the sensitivity. The chief advantages of the method are the simplicity of the apparatus required, the rapidity of the distillation of the arsenic trichloride, and the increased accuracy and sensitivity due to a low reagent blank. This blank may be reduced to zero by the employment of sufficiently pure distilled water.
Acknowledgments The author wishes to acknowledge his sincere appreciation to Leon A. Sweet and C. Kenneth Banks for their valuable assistance. He also desires to thank J. M. Vandenbelt for determining the absorption of the molybdenum blue color of the arsenic solution submitted and for drafting the curve shown in Figure 3.
Literature Cited (1) Bang, Ivar, Biochem. Z., 161, 195 (1925).
(2) Hubbard, D. M., IND.ENG.CHEM.,AXAL.ED.,13, 915 (1941). (3) Morris, H. J., and Calvery, H. 0..Ibid., 9,447 (1937). (4) Rodden, C . J.,J . Research Natl. BUT.Standards, 24, 7 (1940). (5) Sobotka, H., M a n n . W., and Feldau, E., Arch. Derm. Suph., 42, 270 (1940). before t h e Division of Medicinal Chemistry a t t h e 105th Ivfeeting of the AMERICAN CHEMICAL SOCIETY,Detroit, Mich. PRESENTED
