December 15, 1941
ANALYTICAL EDITION
tion is shown in Figure 2. I n determining bismuth in the range 20 to 3000 micrograms, the concentration of potassium iodide should be at least 1 per cent. The Leonard method employs about 0.3 per cent potassium iodide. In this range of potassium iodide concentration a small error in measure ment of the potassium iodide could result in an appreciable error in the bismuth content. The technique here described, if followed precisely, will result in the minimum error due to measurement of reagents. This method bas been employed routinely in the investigation of the bismuth content of biological samples in conjunetion with studies on the absorption and excretion of bismuth by the animal organism originating in these laboratories (7).
915
Literature Cited (1) Bodnar and Karell, Biochem. Z.,199, 2 9 4 0 (1928). (2) Lehrnan, Himulik, and Riohardson, J . Lab. C h . Med., 21, 95-7 (1'335).
(3) Leonard, J . Phrnaeol., 28, 81-7 (1926). (4) Oettingen, von. Physiol. Rea., 10, 221-81 (1930). (5) Rasrnussen, Jackerott, and Sohou, Dansk. Tids. Farm., 1, 391403 mm>. (6) Soott, "Stanhard Methods of Chemical Analysis", 4th ed., p. 79, New York, D. Van Nostrand Co.. 1925. (7) Sondern, Pugh, K d i c h , Lann, and Wiegand, J . Am. Phorm. Assoc., 29. 346-7 (1940). before the Divislon of Analyticd and Micro Chemistry a t the lOlst Meeting oi the American Chcmioul 8ooiety. St. Louis, M o . PRESENTDD
Determination of Arsenic in Biological Material A Photometric Method DONALD M. HUBBARD Kettering Laboratory of Applied Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio
S ' .
TUDIES necessitating the accurate quantitative determination of arsenic in biological material made apparent the need for a rapid and flexible method of analysis by which large numbers of samples, containing arsenic in amounts ranging from less than a microgram to several milligrams, could be handled simultaneously. Recent literature describes a number of methods ( I , 2 , 4 , 6 , 6 ) of acceptable accuracy but none of them filled the author's needs completely. Combination of the better features of these methods plus some slight modifications resulted in the development of a procedure which met all requirements. This modified method consists
essentially of the webashing procedure suggested by Kahane and Pourtoy (4) and later used by Morris and Calvery (6),the evolution of arsenic trichloride from the prepared sample in the manner described by Scherrer (8) and Rodden (7), and a final photometric measurement similar to that described by Morris and Calvery (6).
Procedure
916
I N D U S T R I A L AND E N G I N E E R I N G C H E M I S T R Y
Vol. 13, No. 12
ml. of concentrated sulfuric acid (soecific eravitv 1.84). 20 ml. of TABLE I. ARSENICRANGE,VOLUMES OF DEVELOPING SOLUTION, AND FINAL DILWIONSOF DEVELOPED COLOR MiWW7CZm8
Volume Used MI.
0- 10 0- 50 0-100
5 25 50
Range
and the other. 30 ml. of conceitrated nitric acid (specifii mavitG
Find Dilution M1. 10 50
100
h&e been added, the ma'ior portion of the oraa'& matter Gill have TABLE 11. RECOVERY OF KNOWN AMOUNTS OF ARSENIC ADDED 'TO URINE
the remainder of the nitric acid, reserving 1 ml. in the funnel. Continue heating until dense white fumes of perchloric acid berin
Range Used Micrograms
(50-ml. samples) Arsenic Added Arsenio Found Micrograms Microgiom s
Arsenio Recovered
minutes after the initial formation of copious fumes 07 sulfur trioxide, , (Excessive charring should be avoided by the immediate addition of nitric acid when charring occurs. Excess perchloric acid in the presence of sulfuric acid as used here has not been found daneerous.) Allow the distillation flask with contents to cool, dkonnect and transfer the entire contents to a 50-ml. glassatoppered c;linder, rinsing several times with small portions of distilled nater. Mia well and when cool dilute to the mark at 20" C.
a Cslouisted zs a reagent blank. 50 ml. of distilled water being substituted for 50 ml. of urine.
as-
~~~~~~~~
heating period of approximately 35 minutes). Transfer the tillate plus a few milliliters of distilled water used to rinse down
quat to a 100-ml. beaker, cover with a Fisher Speedyvap beaker cover, and evaporate to dryness. Remove beaker cover and heat the beaker with contents in an electric oven at approximately 120" C. for one hour to remove all trace of oxidizing agents. DEVELOPMENT OF COLOR. Add the correct amount of develop-
minutes will not harm' the d;?veloped color.
[The developing
tilled water. and 1per cent by volume bf a freshl? prepared a q u e
FIQURE 2. DISTILLATION APPARATUS
its fdi int&sity, remove the beaker with contents from the-water bath, allow to cool, and transfer quantitatively to the proper-sized graduated cylinder (Table I), mixing well and diluting to the mark at 20' C. PHOTOMETRIC MEASUREMENT OF COLORDENSITY. Density measurements may be made immediately or 24 hours later. The ~
Transfer one half of ISOLATION OF ARSENIC AS TRICHLORIDE. the prepared sample (25 ml.) by means of the separatory funnel to the distillation flask (200-ml. capacity) used for volatilisation (Firmre 2). In the same manner add 5 ml. of hvdrohromic acid i40"~er &nt) and 40 ml. of concentrated hGdrochloric acid
with R few milliliters of distilied water. Connect B ;arbon droxide
~~~~~
~~
arsenic (as an aqueoussalution of arsenic pentoxide) treated in the s a n e way ss the evaporated and dried distillate residues.
Analytical Results I n Table I1 are listed results obtained by the analysis (in duplicate) of 50-ml. samples of urine containing known added amounts of arsenic, and in Table 111, results from 5gram samples of whole blood. The first two results in the "Arsenic Found" column in each table repredent the actual reagent blanks obtained by substituting equivalent amounts of d i 5
-r
December 15, 1941
ANALYTICAL EDITION
0.80 0 70
0.60
:::p o’T-0.30
0 .o
1 10
2 20
3 4 5 6 7 30 40 50 60
8 70
9 80
1 90
0 A 100 9
MICROGRAMS ARSENIC
FIGURE3. ARSENICCURVE tilled water for 50 ml. of urine and for 5 grams of blood, respectively.
Discussion The size of the aliquots of prepared samples chosen for distillation and color development is dictated by experience. I n most cases one half of the prepared sample may be used if the quantity of arsenic present in the aliquot is not greater than 1 mg., the maximum quantity ordinarily distilled under the conditions outlined above. Aliquots of the distillates also may be used for color development, but the amount of arsenic in the aliquot should not exceed 100 micrograms. If more than this amount is present, the color development should be repeated with a smaller aliquot of the distillate; if this is not possible, the distillation step must be repeated with a smaller portion of ashed sample. I n the latter event, care must be taken to see that 10 ml. of sulfuric acid (specific gravity 1.84) are present during the distillation. I n the case of certain materials, particularly blood and spinal fluid, usually low in arsenic, and when only very small samples are available, the entire sample is used for the analysis. The technique of wet-ashing employed reduces to a minimum the amount of arsenic lost by entrainment in the distillate, in confirmation of Morris and Calvery’s (6) work. A definite excess of nitric and perchloric acids is maintained a t all times during the destruction of organic matter, and entrainment losses are negligible as compared with the sensitivity of the final photometric measurement. A separate analysis of the distillate from the wet-asking of 100 ml. of urine containing 4000 micrograms of arsenic showed a loss of only 12 micrograms or 0.3 per cent of the original content. The blue color produced in the conversion of arsenic acid to an arsenic-molybdenum compound by treatment with a solution containing hydrazine sulfate and ammonium molybdate has been found to be very stable; density measurements have remained constant over a period of 24 hours, obeying Beer’s law; this confirms the findings of Rodden (7). The temperature of the water bath used in color development may range from 70” to 75’ C. Constant temperature has proved to be unnecessary. The distillation step separates the arsenic from silica and phosphorus, which also give blue-colored
917
molybdenum compounds, while other volatile chlorides of antimony, selenium, and germanium do not interfere in the subsequent color development. It is essential to run a reagent blank for each lot of reagents used, because of their variability in arsenic content (Tables I1 and 111). Dust and other particulate matter such as cigaret ashes contain small amounts of arsenic; therefore it is necesary to guard carefully against dust contamination, particularly when dealing with small quantities of arsenic. The use of battery-type equipment with adjustable electrical heating facilities makes possible the uniform, rapid, and convenient handling of a large number of samples daily in a small working space. What is more, after the digestion process has started, it requires little attention. The use of interchangeable joints a t strategic places in the distillation apparatus also makes the operation more convenient, particularly in respect to rapid cleaning. The accuracy of recovery by this method is superior to that of the method described by Chaney and Magnuson ( I ) , and equal to that obtained by Morris and Calvery (6), but in the author’s hands, a t least, the procedure seems to be more rapid and the substitution of the distillation step for the arsine evolution and combustion technique makes for simplicity as well as compactness of apparatus. A further advantage lies in the fact that a single pure color is employed, instead of the “mixed color” obtained by Chaney and Magnuson (1). The method has been found applicable to a wide variety of biological materials and has been especially useful in the analysis of the normal daily fecal and urinary excretions of rabbits, which entails an accurate determination of arsenic in amounts of less than 10 micrograms, and in the analysis of small samples of blood and spinal fluid. Several modifications would aid in accelerating the analyses in certain cases-for example, when extremely large amounts of arsenic are present, the distillate may be titrated directly by Scherrer’s method (8). Again, the possibility of determining arsenic polarographically is being investigated in this laboratory, and the preliminary work promises a sensitivity comparable to that of the method here reported, with the advantage of increased simplicity. It is hoped that the data on this development will be available shortly.
TOLE111. RECOVERY OF KNOWNAMOUNTS OF ARSENICADDED TO
WHOLEBLOOD
(5-gram samples) Arsenio Added Arsenio Found Micrograms Micrograms Nil 0.5a Nil 0.7a Nil 0.6 Nil 0.7 1.0 1.7 1.5 1.0
Range Used Micrograms 0- 10 0- 10 0- 10 0- 10 0- 10 0- 10 5.0 0- 10 0- 10 5.0 0- 50 15.0 15.0 0- 50 50.0 0- 50 50.0 0- 50 0-100 100.0 100.0 0-100 a Calculated as reagent blank, 5 grams of for 6 grsms of whole blood.
5.5 5.7 16.0 15.5 50.5 50.0
100 99
Arsenio Recovered Micrograms
...
1.1
0.9 4.9
5.1 15.5 15.0 50.0 49.5 LOO 99
distilled water being substituted
Summary A photometric method has been applied to the microdetermination of arsenic in small samples of biological material-for example, 100 ml. or less of urine and 10 grams or less of blood or other tissue. The sample is prepared for analysis by the wet-ashing method, employing perchloric, nitric, and sulfuric acids. The arsenic contained in the ashed sample is isolated completely
INDUSTRIAL AND ENGINEERING CHEMISTRY
918
from other interfering constituents, such as silica and phosphorus, by the in a apparatus designed to ensure ease of cleaning and charging. The arsenic is oxidized to arsenic acid and the density of the blue color obtained b y formation of the stable arsenicmolybdenum compound is measured photometrically. Three ranges are used for the quantitative estimation of the arsenic-namely, 0 t o 10, 0 t o 50, and 0 to 100 m i c r o g r a m s the accuracies for the above ranges being, respectively, * O , l , +0.5, and *l.O micrograms. A reagent blank which varies from 0.5 t o 2.0 micrograms is run simultaneously with each set of analyses and i t is determined with a n accuracy of *0.1 microgram, thus securing an accuracy Of *OS2 microgram for the lowest range.
Vol. 13, No. 12
Literature Cited (1) Chancy, A. L.,and Magnuson, H. J,, IN=. EXG.CHEM.,ANAL. ED., 12, 691-3 (1940). (2) Hinsberg, K., and Riese, M., Biochem. Z.,290, 39-43 (1937). ANAL.ED.,12, 768-71 (1940). (3) Hubbard, D. M., IND. ENG.CHEM., (4) Kahane, E., and Pourtoy, M., J . pharm. chim., Ser. 8 , 23, 5 (1936). ( 5 ) Maechling, E. H.,and Flinn, F. B,,J . Lab. Clin. &fed., 15, 779 (1930). (6) Morris, H. J., and Calvew, H. O.,IND.ENQ.CHEW,ANAL.ED., 9, 447 (1937). (7) Rodden, C. J., J . Research Natl. Bur. Standards, 24, 7 (1940). (Research Paper 1267.) (8) Schemer, J. A., Ibid., 16 (1936). (Research P a p e 871.) P R ~ S E N Tbefore E D the Division of Analytical and Micro Chemistry at the 102nd hleeting of the American Chemical Society, Atlantic City, N. J.
Apparatus for Extraction of Lipoids from Wet Tissues FREDERIC E. HOLMES Children’s Hospital Research Foundation and Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio
T
HE small apparatus shown in Figure 1 has been used in
this laboratory since 1932 for the extraction of lipoids from blood in the determination of cholesterol. The method used has been published only in the abstract (8) without a sketch of the apparatus. The essential modification in this extractor from previous ones of similar type, such as those of Leiboff (19, IS), Ling ( 1 4 , and numerous ot,hers.is the trap fixed to the reflux condenser at its lower end. Moisture from the sample distills with the solvent and is retained in the trap. A notable improvement in consistency of results has been obtained, and there are reasons for concluding that direct extraction with chloroform under these conditions results in complete, or nearly complete, removal of cholesterol from the sample. However, although most of the author’s work has been done with chloroform as the solvent, this extractor, and the larger ones shown in Figures 2 and 3, are adaptable to use with other solvents.
Microextractor The apparatus (Figure 1) consists of an extraction tube having bulb at the bottom calibrated to contain 5 ml. of extract and four lugs to support the sample, and a separate one-piece assembly of condenser and tra which fits into the top part of the tube. The body of the t u i e above the constriction is of 2 4 to 25-mm. tubing and has an approximate length of 230 mm. The four lugs, shown laterally and in section, are approximately 50 mm. above the top of the constriction. The constricted part is of 11-mm. tubing and has a length of 13 to 15 mm., these dimensions having been found best to give sufficient accuracy in making up to volume and still to permit rapid and thorough mixing of the extract with the reagents in the determination of cholesterol. The condenser has a long skirt which serves to hold it and the trap in the center of the extraction tube and also to deflect any moisture from the air down the outside of the a p paratus. The extraction tubes and condenser skirts are kept within close enough tolerances so that a large number of tubes and eeveral condensers are interchangeable. The outer shell of the trap and the part of the condenser hanging in the extraction tube are of 16-mm.tubing. The inner tube of the trap is of 11-mm. tubing extending a t least 12 mm. below the ring seal joining it to the shell, and the shell extends like a collar at least 12 mm. above the seal. The openings through the sup orting member connecting the trap to the condenser have a Zameter of not less than 5 mm. The holes to the outside through the shell below the ring B
seal have approximately 3 mm. diameter. The bottom of the trap should be between 15 and 30 mm. above the lugs. For operation, 5 to 6 ml. of solvent are placed in the bulb, a piece of fat-free filter paper 20 X 20 X 1 mm. with its corners bent at right angles is dropped ‘‘ears up” onto the lugs, 0.25 ml. of the specimen (serum, h,ep a r i n plasm a , whole blood, “saline” suspension of cells, o i other fluid) is delivered onto the paper from a graduated 1-ml. pipet or from a special pipet ( 7 ) , and the extraction tube with the condenser in p l a c e is i m mersed to the level of the sample in a boiling water bath. (Atall-form 400-ml. Detail beaker makes a f Trap satisfactory bath for two tubes.) The extraction is continued for 1 hour. Vapor of the boiling solvent around and t % ? ~ ~ ~ the sample, carrying moisture from J J the sample to the - S e c t i o n Ir, condenser. T h e condensate flows into the trap through the openings in the sup-
6 8
8 T : : : ;
separate in the inner tube of the trap, the water beinn
vntn;nnA
anrl
th; solvent flowing around the bottom of the inner tube
FIG^ 1. MICROEXTRACTOR FOR EXT~ACTION OF LIPOIDS FROM BLOOD Condenser and trap in position in tube
