Colorimetric Microdetermination of Arsenic ALBERT L. CHANEY’ AND HAROLD J. NAGNUSON College of Medical Evangelists and University of Southern California School of Medicine, Los Angeles, Calif.
I
T HAS been found possible to shorten and simplify the
colorimetric determination of arsenic on biological material by the use of a new distillation procedure. An important feature of this procedure is a constant-volume distillation by means of a recently developed still ( 2 ) . The arsenic is distilled as arsenic trichloride after acid digestion of t’he original sample. The preliminary digestion of the material and the final colorimetric determination are similar to the procedure described b y Morris and Calvery ( 7 ) , Maechling and Flinn ( 6 ) , and others (1, 3, 4,6, 8,9). The following modifications are introduced into the intermediate isolation of arsenic: The distillate is used directly for color development without evaporation with nitric acid, or other treatment. The special still makes possible a 5-minute distillation and a minimum volume of distillate. The hydrochloric acid accompanying the arsenic trichloride is used to control the acidity in the color determination. Arsenic is oxidized to the pentavalent state in the distillate by the use of a small known excess of potassium iodate.
The use of this procedure has necessitated some changes in the reagents during digestion and color development as well. The colorimetric determinat’ion of arsenic has obvious advantages of accuracy and convenience over the Gutzeit and similar procedures. I n addition, the contamination of laboratory apparatus by heavy metals such as lead and mercury is avoided. The method is most useful in the range of 1 to 100 micrograms of arsenic.
head is available from K. D. Johnson, 1115 Arroyo Verde, South Pasadena, Calif.) Heater, 350 watts (Cenco “hot cone”), variable transformer (General Radio Variac or eauivalent). uhotoelectric colorimeter. and tubes graduated at 25 cb. , I
Procedure DIGESTIOS.From 5 to 20 grams of tissue or 5 to 100 cc. of urine are placed in the digestion flask. To the sample are added 10 cc. of concentrated nitric acid, 5 cc. of concentrated sulfuric acid, and small pieces of alundum as boiling stones. The flask is connected by means of a U-tube to the condenser, as shown in Figure 2, and a second flask wit,h attached soda-lime tube is connected to the bottom of the condenser. With this arrangement digestion may be carried on in the laboratory without the presence of objectionable fumes and no hood is required. The flask is heated on the electric heater with reduced voltage (about 60 volts) while the digestion is proceeding. The variable transformer provides a convenient method of heat control. Nitric acid is added t,hrough the dropping funnel from time to time t o prevent charring. When the digestion is nearly complete, as shown by the slightly yellow color of the solution, 0.5 cc. of SO per cent perchloric acid is added to clear the solution and complete the digestion. After cooling sufficient’ly,the contents of the digestion flask are diluted with 10 to 15 cc. of water, added through the dropping funnel. The diluted digest is boiled vigorously, using full voltage on the electric heater, until all the perchloric acid is driven off and strong fumes of sulfuric acid are evolved. After cooling, the specimen is ready for distillation.
FIGURE1 SPECIAL STILL
Reagents Concentrated sulfuric acid, arsenic-free, reagent grade. Concentrated nitric acid, arsenic-free reagent grade. Perchloric acid, 60 per cent, reagent grade. Reducing reagent: 5.0 grams of hydrazine sulfate, 17.0 grams of potassium chloride, and 4.0 grams of potassium bromide per 100 cc. of solution. Potassium iodate, 0.3 per cent. Ammonium molybdate, 0.5 per cent (should not be kept over 1 week). If necessary, ammonium molybdate may be recrystallized from hot water to reduce phosphate content. Hydrazine sulfate, 0.15 per cent. Standard arsenic solutions. Dissolve 1.319 grams of reagent grade arsenic trioxide in 15 cc. of 1 N sodium hydroxide. Dilute, add 15 cc. of 1 S hydrochloric acid, and make up t o 1 liter. This contains 1 mg. of arsenic per cc. Dilute standards of 0.05 and 0.01 mg. per cc. are prepared from this stock solution. Apparatus Distilling flasks, two necks, round bottom, 250-ml. capacitv with standard taper joints (24/40 center neck, 19/38 side neck?. Dropping funnel with 19/38 standard taper joint t o fit 250-ml. flask. Reflux condenser, Graham, coil type, 200 mm. in length with a 24/40 standard taper joint a t top and bottom. Special still shown in Figure 1. Somewhat similar to a Soxhlet extractor in appearance, the stillhead, a, is fitted with standard taper joints (24/40) at each end for connection with the twonecked distilling flask and reflux condenser. A small lip is sealed in below the end of the drip tip of the condenser to direct the reflux down the capillary tube, which should be about 1 mm. in diameter and approximately 10 em. in length. Just below this is a baffle plate, b, to prevent splashing into the capillary return. A second baffle plate, b’, is used just above the flask to prevent mechanical entrainment of spray from the distilling flask. The trap, t, has a series of indentations t o provide more effective washing of steam and absorption of arsenic. (The special distilling 1 Present
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address, Lo8 Angeles County General Hospital, Los Angeles, Calif.
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the intensity of the color. Standards containing 0.01 or 0.05 mg. of arsenic are prepared in the same manner. To each standard are added 5 cc. of 1 N hydrochloric acid and 0.3 cc. of potassium iodate.
Experimental Data Table I shows the recovery of arsenic by distillation from reagents only. Six determinations were made a t each level. The mean of these determinations is shown, together with the average deviation from the mean. The arsenic was added in pentavalent form to the reagents and the color comparison was made with similar quantities of arsenic added to 5 cc. of 1 A' hydrochloric acid and the color reagents added directly. Table I1 shows recovery of arsenic in various forms added to tissues before digestion.
Discussion DIGESTION.During digestion care should be taken that charring does not occur, in order to avoid loss of arsenic. Since nitrate causes fading of the final color, and perchloric acid interferes with its development, it is necessary to remove these interfering substances completely before distillation. This is best done by adding water and boiling to strong fumes of sulfuric acid. DISTILLATIOS.The special distillation apparatus functions in the following manner:
4
cm.
FIGURE 2. DIAGR.4M
OF
APPARATUS
DISTILLATION.Five cubic centimeters of water are added to the sulfuric acid digest in the flask. The dropping funnel, f, containing 2.0 cc. of the hydrazine sulfate, potassium chloride, and potassium bromide solution is connected to the side neck of the flask, as shown in Figure 1. The flask is connected to the still by the center neck and then placed on the hot 350-watt heater. After boiling has started and steam has started to condense in the trap, 3 cc. of 0.3 per cent potassium iodate solution are added through the top of the still into the trap. The reflux condenser is put in place at the top of the still, so that the reflux will drip down the capillary tube. The contents of the dropping funnel are blown into the flask and washed down with 2.0 cc. of water. Distillation is continued about 4 to 6 minutes, until iodine vapors condense into crystals in the reflux condenser. The flask and the still are disconnected and the contents of the trap are poured through the top into the 25-cc. graduated tube. The trap is rinsed out two or three times with 3 cc. of Tyater, so that the final volume in the tubes is from 15 to 17 cc. The distillate should be faintly yellow, owing to presence of free halogen. The arsenic in the distillate will have been oxidized by the potassium iodate to the pentavalent state. As an alternative distillation procedure, distilled water may be added t o the trap in place of the potassium iodate solution, and the distillation carried out for 4 to 5 minutes after the addition of the reducing mixture. In this case oxidation of the arsenic is carried out by adding 0.2 cc. of the potassium iodate to the distillate in the 25-cc. graduated tube. This procedure should be used only when any losses of sulfuric acid during digestion are made UP. so that fullv 5 cc. of sulfuric acid are present in the distilling 'flask. The graduated tube is placed in a COLORDEVELOPMENT. boiling water bath and 2 cc. of 6.5 per cent ammon'um molybdate are added, followed by 2 cc. of 0.15 per cent hydrazine sulfate. The heating is then continued for 10 minutes. After cooling and dilution to 25 cc., the color is read in a photoelectric colorimeter using a 725 filter and either a 1- or 2-em. cell, depending on
The steam evolved from the still bubbles through trap t (Figure l), xhich contains water. The hydrochloric acid and arsenic trichloride accompanying the steam are thereby retained in the trap. Significant losses do not occur, since the solution is not only dilute but lower in temperature than the contents of the distilling flask. When potassium iodate is present the arsenic is oxidized almost immediately to the pentavalent state and thereby rendered nonvolatile. The steam, after being washed by the contents of the trap, is condensed and returned t o the still by the lip and capillary return. In this way the volume of distillate remains very small and the change in concentration of the contents of the distilling flask is minimized. The volume of distillate in the trap is increased somewhat by heat losses and resultant condensation of steam, but this amounts to only 5 cc. in a 10minute period. The function of the lower baffle plate, b', is to prevent mechanical carry-over of appreciable quantities of phosphate. When materials which are very high in phosphate, such as bone, are digested, a considerable blank due to phosphate will be present even when using b'. In such cases the interference of phosphate may be practically eliminated by a double distillation-i. e., the distillate after oxidation with iodate is evaporated with 5 cc. of concentrated sulfuric acid, and then redistilled.
OF BRSEXIC FROY REAGENTS TABLEI. RECOVERY
Arsenic Added Micrograms Sone
Arsenio Recovered Microgram8 0.1 1 . 1 5 t 0.06 2 . 1 * 0.12 4.2 = 0.07
1 2 4
5.7 7.9 10.2 28.9 49.4 104 0
G 8 10 30 50 100
f
Recovery
%
iij 105 104 95
0.22
* 0.24
99
loa
t 0.6 += +
96 99
0.7 0.9 2.8
104
O F .%RsENICF R O M TISSTJES TABLE11. RECOVERY
Arsenic Added M~crog~ams None 10 50 100 10 30 50 100 8.3 42.3 96.0
Form
as;6;* Aslo6
As206 Xapharsen Mapharsen Sfapharsen Mapharsen
Seoarsphenamine Neoarsphenamine Seosrsphenamine
Arsenic Recovered Micrograms 0-0.8 11.2 45.8
101.5
8.7 24.8 45.0 10'2.6 7.2 46
90.6
Recovery
%
...
112 9'
101 5 87
83
00 102.6 93 109 94
NOVEMBER 15, 1940
AIVALYTICAL EDITION
COLOR DEVELOPMEKT. The relative quantities of molybdate and acid present must be maintained within close limits (6, 6, 8, 9). At low acidities, color develops in the blanks, largely owing to silica. Too great acidity markedly decreases the intensity of the color. The optimum ratio is about 5.0 cc. of 1 N hydrochloric acid to 2.0 cc. of 0.5 per cent ammonium molybdate a t a volume of 20 cc. Ammonium molybdate was found definitely superior to the sodium molybdate, since the former allowed a somewhat wider range of acidities without change of color intensities. It is also important to develop the color a t the correct volume in order to maintain the acidity in the proper range. I n harmony with other investigators, the authors feel that hydrazine sulfate is the most suitable reducing agent for developing the color (6, 7 ) . More hydrazine sulfate than that previously recommended is used in order to reduce the excess potassium iodate used in oxidation of the arsenic. Ten minutes’ heating in a boiling water bath is sufficient t o develop the color, which is then stable for several hours. A blank should be run on distillate from sulfuric acid alone, and should correspond to less than 0.5 microgram of arsenic. Either a 660-millimicron or 725-millimicron filter may be used for reading the color, though the latter is preferable. The very slight yellow color due to excess hydrazine sulfate does not interfere a t these wave lengths. Between 1 and 100 micrograms the color intensity follows
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Beer’s law within 2 per cent. A 2-em cell is used up to 50 micrograms and the 1-em. cell for quantities of 50 to 100 micrograms. IKTERFERISG SUBSTANCES.Antimony, germanium, and selenium may accompany arsenic Tvhen distilled with hydrochloric acid, but do not give color with molybdenum reagents under the conditions described. Phosphate, which reacts like arsenate m-ith the molybdenum reagent, is nonvolatile and is thus prevented from interfering. Kitrate and perchlorate interfere with color development and should be removed prior to distillation.
Literature Cited Assoc. Official Agr. Chem., “Official and Tentative Methods of Analysis”, p. 372, 1935. Chaney, A. L., ISD. EXG.CHEM.,Anal. Ed., 12, 179 (1940). Deemer, R. B., and Schricker, J. A,, J . Assoc. Oficial Agr. Chcm., 16, 226 (1933). DenigBs, G., Compt. rend.. 171, 802 (1920). Kuttner, T., and Cohen, H. R., J . B i d Chem., 75, 517 (1927). Maechling, E. H., and Flinn, F. B., J . Lab. Clin. Med., 15, 779 (19301. Morris, ’H. J., and Calvery, H. O., IND.ENQ.CHEM.,Anal. Ed.. 9, 447 (1937). Truog, E., and Meyer, A. H., Ihid., 1, 136 (1929). Zineadze, C., Ihid., 7, 230 (1935). THIS work was done b y H. J. Magnuson on a research fellowship of t h e American College of Physicians.
Removal of Static Charges from Glassware by Ultraviolet Light CLERIENT J. RODDEN, National Bureau of Standards, Washington, D. C.
THE
and distance of the ultraviolet light source are given in Table I. The vessel was 15-iped with a dry chamois and was considered to have reached constant weight when the variation was less than 0.05 mg. using a macrobalance. Weighings were made usually after exposure to the light a t 5-minute intervals. I n some instances this was reduced to 2-minute intervals For general practice i t is recommended that the wiped article be placed about 60 cm. (2 feet) from either a Hanovia Alpine sun lamp or General Electric Lab-Arc, for 10 minutes.
static charge acquired by glassn-are when wiped may cause a serious error in weighing the vessel. In the winter months, when the moisture content in the atmosphere is low, this difficulty is more apt to be encountered. In the Pregl (1) method for the microchemical determination of carbon and hydrogen the absorption tubes are wiped with a chamois. This causes a static charge which does not leak off soon enough, when the humidity is low, to maintain the time schedule (usually 10 to 15 minutes) between completing a combustion and weighing the absorption tubes. Van Straten and Ehret ( 2 ) state that the charge can be removed by the high-frequency discharge from a device commonly used to detect pinholes in evacuated apparatus. The author has found that if ultraviolet light is used to ionize the air surrounding a glass vessel, the charge can readily be dissipated. D a t a on the effect of time of exposure
Literature Cited (1) Pregl, F., “Quantitative Organic hdicroanalysis”, 2nd ed., p. 46, Philadelphia, P. Blakiston’s Son & Co., 1930. (2) Van Straten, F. W., and Ehret, W. F., IND. ENQ.CHEM.,Anal. Ed., 9, 443 (1937).
TABLE I. EFFECT OF TIMEOF EXPOSURE AND DISTANCE OF ULTRAVIOLET LIGHTSOURCE Vessel
Relative Humidity
% Pyrex tube, 10 mm. X 11 cm. 150-ml. Erlenmeyer flask (Pyrex)
Temperature O
c.
40
75
29 28 2s 34 23
70 70 76 73 73 76 80
23
SO
29 38
Increase in Weight Caused by Wiping Mg.
20 5
Source of Ultraviolet Light
Hanovia Alpine sun lamp, 110 volts, alternating current
18
General Electric Lab-Arc, IlOvolts, direct current
10
G-5 in special envelope, General Electric
Distance from Source Feet 2
Time Required for Discharge Min. 10
1 3 2
10 10
5
8 10
3 1
10 10
Still changing in wgight after 20 min.
