V O L U M E 26, NO. 3, M A R C H 1 9 5 4
595
Table I. Results of Test Procedure Areenic Added, Mg.
Arsenic Found, Mg.
Antimony Added. 31g.
Antimony Found, Mg. 1.020 1.140 0.800 0.152
EXPERIMENTAL RESULTS
The procedure was tested by generating arsine and stibine from 6JI sulfuric acid solutions containing tripositive arsenic and antimony by treatment with zinc. This resulted in some loss of arsine when more than 0.100 mg. was evolved. There is mme question as to the completeness of conversion of antimony to stibine under these conditions although over 90% rerovery was observed in samples containing over 1 mg. of antimony. Results of the test procedure appear in Table I. CONCLUSIONS
-0 5 7
L
I
I
-060
-063
Ey2
vs
Larger quantities of arsenic and antimony could be handled by the use of larger absorption tubes. The procedure has been used to determine traces of arsenic and antimony in various materials at the Xaval Research Laboratory.
1
-0 66
S C E
LITERbrTURE CITED
Figure4. Effect of ( H + ) o n Ei/,vs. of ArsenicArsine Wave 0 El/, V S . log molarity of H’ in 2 M C10
( 1 ) Banibach, K., INn. EX. CHEY.,.ISAL. ED.,1 4 , 265 (1942).
( 2 ) Kolthoff, I. lI.,and Iingane. .J
El:, 1‘s. log molal activity of HCl (no inert salt)
(3) (4) (5)
solution to be polarographed is allowed t o stand for long periods exposed to air. Sulfur dioxide gives a wave in this medium which interferes with the first arsenic wave. However, the heatin of the solution during the extraction plus the usual removay of oxygen with nitrogen gas prior t o polarographing the solution is adequate treatment for complete removal of sulfur dioxide. The solution containing extracted tripositive antimony and arsenic is polarographed after making up to known volume (10.0 ml.) and bubbling nitrogen through for 10 minutes.
(6)
(7)
“Polarography,” Yew I-ork, Interscience Publishere, 1941, 2nd ed., 1952. Lingane, J. J., IND. E s c . C m w , ASAL.ED.,15, 583 (1943). Lingane, J. J.,and Laitinen, H. .1., Ibid., 11, 504 (1939). Sandell, E. B., “Colorimetric Determination of Traces of Metals,” pp. 136-43, Sew York, Interscience Publishers, 1944. Snell, F. D., and Snell, C . T., “Colorimetric Analysis,” Sen. York, D. Van Nostrand Co., 1936. Webster, 9. H., and Fairhall, C’. T.. .J. Ind. Hug. To.~icoZ.,27, 183 (1945).
RECEIVED for review December 18. 1952. Accepted November 23, 1933. Presented in p a r t before t h e Division of Physical a n d Inorganic Chemistry at the 123rd Meeting of the - 4 4 1 ~ ~ CHEMICAL 1 ~ ~ s SOCIETY, Los Angeles, Calif.
Determination of Arsenic in Biological Materials ROBERT JOHN EVANS and SELMA L. BANDEMER Department of Agricultural Chemistry, M i c h i g a n State College, East Lansing, M i c h .
METHOD is described
for detprmining the arsenic content of various biological materials, such as eggs, fresh and dried live1 and muscle tissues, skin, and similar materials. 1
Determination of the arsenic content of eggs is especially difficult because of the very small amount present and of the high lipide content of eggs. Oxidative digestion with nitric, sulfuric, and perchloric acids is slow, especially when samples of eggs used are large enough t o give fairly accurate arsenic determinations. .4n attempt was made to apply the hydrochloric acid digestion procedure of Kingsley and Schaffert (8) to eggs and fresh chicken livers, but recoveries of added arsenic trioxide were always poor. The procedure of Bandemer and Schaible ( 2 , 3) for ashing eggs \\-as adapted for use with eggs and fresh and dried animal tissues so as t o produce an ash with no loss of arsenic. Arsenic was distilled as arsine and the color was then developed and read with a spectrophotometer as described by Kingsley and Schaffert
The homogenized liquid egg contents were mixed with saturated magnesium nitrate solution in a filter paper-lined crucible, charred over a hIeker burner, and ashed overnight a t 600” C. in a muffle furnace. The finely minced fresh tissues or finely ground dried materials were mixed m-ith magnesium nitrate solution in a crucible lined a i t h filter paper and vegetable parchment paper, evaporated to dryness on the steam bath, slowly ignited in the muffle furnace, and ashed overnight a t 600” C. The ash was dissolved in dilute hydrochloric acid and the arsenic was distilled fiom the ash as arsine, which was collected in a n iodine solution. The arsenic content of the solution was determined by developing the heteropoly blue compound of arsenic and reading the color with a Beckman Model B spectrophotometer with an 840 mp wave length light source.
(8).
Good reroveries of arsenic added to the egg homogenate and either fresh or dried liver tissue were obtained, and the values were reproducible. The method was applied to eggs, to chicken liver, breast muscle, leg muscle, and skin, and t o dried pig tissues. The magnesium nitrate ashing procedure has the advantage over the wet-oxidation method that it is more rapid and does not require large volumes of nitric acid necessary t o oxidize such biological materials by the &-etmethod.
Reagents. SATURATED > ~ G N E S I U MSITRATE SoLmiox. Dissolve magnesium nitrate hexahydrate in warm distilled water by stirring until no more will go into solution. Allow to cool t o room temperature. The solution should always be in contact with crystals of undissolved magnesium nitrate. HYDROCHLORIC ACID SOLUTIONFOR DISSOLVING EGGASH. Mix 175 ml. of concentrated hydrochloric acid with 280 ml. of distilled water and allow t o cool before using.
METHOD
ANALYTICAL CHEMISTRY
596 PoTAssInr IODIDE SOLKTIOS. Prepare a 15yo solution in distilled water just before using. STANNOUS CHLORIDESOLUTION.Prepare a 40y0 solution in distilled water just before using. MOSSYZINC. Use a low arsenic zinc. LEADACETATE SOLUTION.Prepare a saturated solution. STOCKIODISE 0.02A' SOLUTION.Dissolve 2.54 grams of iodiric and 8 grams of potassium iodide in 25 ml. of distilled water and dilute to 1 liter. Store in a dark bottle. 0.001N IODISE S o ~ n i ~ o uDilute . 5 ml. of stock solution to 100 ml. just before using. AMMONILWMOLYBDATE SOL~TTIOS. Dissolve 5.0 grams of ammonium molybdate tetrahydrate in a warm mixture of i o ml. of concentrated sulfuric acid and 300 ml. of distilled water, cool, and dilute to 500 ml. HYDR.4ZINE SuIIFdrrESOLUTIOX.Dissolve 0.3 gram of hydrazine sulfate in ahnut 150 ml. of distilled water and dilute to 200 ml.
Add 6.0 ml. of saturated magnesium nitrate solution and mix well with a stirring rod. Evaporate to dryness on the steam bath. Ignite in a muffle furnace by putting in the cold furnace, slowly raising the temperature to 600" C., and heating a t this temperature overnight. Wet the ash from the eggs or other biological material with water and dissolve it in 17 ml. of the hydrochloric acid solution. Transfer the solution into a 125-ml. Erlenmeyer flask with a 24/40 standard-taper joint. Kash the crucible out with two 12.5-ml. portions of the dilute acid and transfer the washings to the Erlenmeyer flask. Add enough hydrochloric acid in this way to dissolve and neutralize the magnesium oxide formed on ashing and to add the 10 ml. of concentrated hydrochloric acid recommended by Kingsley and Schaffert (8) for distilling the arsenic. Carry out the remainder of the procedure essentially as described by Kingsley and Schaffert (8) with slight modifications. The distillation apparatus was made according to their specifications by Corning Glass Works. To the solution in the Erlenmeyer flask add 2 ml. of 15% potassium iodide solution and 0.5 ml. of stannous chloride solution. Allow to stand for 15 minutes. Add several pieces of mossy zinc and attach to the distillation apparatus. Allow the generated gas to bubble for 60 minutes into 5.0 ml. of 0.001S iodine solution in a test tube immersed in cracked ice. Remove the collection tube. Add 0.5 ml. of ammonium molybdate solution and 0.2 ml. of hydrazine sulfate solution, mixing after each addition. Place in the steam bath for 10 minutes. Cool and read in 1-cm. borosilicate glnss cells with a Beckman Model B spectrophotometer at i i xave length of 840 mp and balanced a t 100% transmittance against n blank, which has been carried through the ashing, distillation, and color development procedures in the same way as the unknown samples for each run. Read the arsenic content from a curve preparcd by adding standard arsenic trioxide solution to Erlenmeyer flasks to give levels of 1 to 107 of arsenic per flask. To each flask add 35 ml. of distilled water and 10 ml. of concentrated hydrochloric acid, distill the arsenic, develop, and rc:td thc color as tlescribctl :hove. WAVE LENGTH OF MAXIMUM ABSORPTIOh
WAVE LENGTH, MILLIMICRONS
Figure 1. Absorbance (Optical Density) of Heteropoly Blue Compound of Arsenic The color was formed by the reaction of a mixture of 5 ml. of 0.001N iodine solution containing the indicated weight of arsenic, 0.5 ml. of 170ammonium molybdate solution in 5N sulfuric acid, and 0.2 mi. of 0.1570 hydrazine sulfate solution. The color was read in a 1-om. borosilicate glass cell with the Beckman Model B spectrophotometer A . Iodine solution contained 107 of arsenic added as A9102 B . Iodine solution contained 5 y of arsenic addod as As& C. 107 of arscnic distilled as arsine into iodine solution D . 57 of arwnic distilled as arsine into iodine solution I
STOCKARSESIC SOLUTIOS. Dissolve 0.1320 gram of arsenic trioxide in 50 ml. of distilled water containing 0.5 mi. of i o r ; sodium hydroxide solution. Neutralize with ionc sulfuric acid solution and dilute to 100 ml. (1.0 ml. contains 1.0 mg. of arsenic). STA~DAR .~R DS E S I C SoLuTIon. Dilute 1.0 nil. of the standard stock solution to 100 ml. (1.0 ml. contains 10.07 of arsenic). Procedure for Eggs. Fold a disk of 12.5-cm. diameter No. 40 Rhatman filter paper as for filtering, fold the tip of the cone back, and fit the truncated cone into a Yo. 4 Coors porcelain cruciblr (3): Homogenize the egg contents in a Waring Blendor. To the weighed paper and crucible add 20 ml. of egg homogenate and weigh, Smaller samples of egg must be used if the eggs contain more than 0.57 of arsenic per gram. Add 6.0 ml. of saturated magnesium nitrate solution and mix well with a stirring rod. Kipe the stirring rod with a small piece of filter paper and add to the crucible contents. Heat over ~tbleker burner a t full heat until the egg has coagulated and charred. Then tritnsfrr to :L muffle furnace and heat a t 600" C. overnight. -1light white :is11 is obtained. Procedure for Other Biological Materials. Fold a disk of 12.5cm. diameter vegetable parchment paper (obtained in sheets from the Weissinger Paper Co., Lansing, Mich., and disks cut by the authors) as described and fit into the cruciblr Then fold :L similar disk of No. SO Whatman filter paper and fit inside thr parchment disk. Weigh into the filter paper from 5.0 to 20.0 grams of fresh ground tissue or 0.2 to 10.0 grams of dried finely ground biological material, the amount depending upon the arscnic content. I t should not contxin more than 107 of arsenic.
The heteropoly blue compound formed by condensation of aracnate arid molybdate ions to form molybdiarsenic acid which is reduced by hydrazine sulfate to the stable blue compound has been uscd for many years in the determination of arsenic (4-19). Early Tvorkeis (6, 7 , 9, 11, la) measured the transmittancy at 610 to i 2 5 nip wave length, probably because of the limited range of their photoelectric colorimeters, but Sultzaberger (15') using a Beckniaii spectrophotometer found that maximum absorption for the arsenic complex is in the infrared a t about 840 mp. Most of the later m-orkers have used the 840 mp wave length for reading the arsenic color ( 4 , 6, 1 0 ) . Kingsley and Schaffert (8) used 865 mp light transmission with the Beckman Model DU spectrophotometer and 830 mp with the Coleman Model No. 14 spectrophotometer. Absorbancy curves werc obtained with the Beckman Model I3 spccstrophotometer using the procedure for obtaining a standardization curve described by Kingsley and Schnffert (8) and also by the method herein described for the distillation of known quantities of arsenic trioxide, The curves obtained are given in Figure 1. In each case maximum absorbance or minimum transmittance \vas obtained a t 840 mp. This particular instrument had been checked with a didymium glass filter, using the wave lengths of maximum absorption obtained at the Sational Bureau of Standarc19 ( 1 2 ) . DISTILL iTIOh OF ARSEYIC
Completc recovery of clistilled arscnic was not obtained, but recovery was constant a t 87 to 00%. For this reason standard curves were prepared by distilling known quantities of arsenic trioxide : t ~described in the methods. .4 typical standardization curve is presented in Figure 2. Curve A was obtained by making the readings with the 865 mp light snurce and curve B by reading the same solutions with the 840 mp light source. The indicated number of micrograms of arcenic were added to the distillation flasks and the distillation was carried out as described under methods. Figure 2 shows that the standard curve obtained differs from the wave length of light source used or, in other words,
V O L U M E 26, NO. 3, M A R C H 1 9 5 4 Tahle I.
597
Recorery of Arsenic Trioxide Added to Eggs before Ashing (IO-, of arsenic added as Asz03)
l l e t h o d of Ashing
Amount of bIg(N0a)S Eoln. Added, All.
Weight Eggs, Grams
Recovery oi As203 Added t o Eggs. 7 6
1.5
10
06
1.5
10
20
1.j
10
67
1 .5
10
23
1 ..i
25 23 23 20 20 20
3.j 45
lilagncsirirn chioride added before charrinr, 3Iagneaio?ii riiloride added after charring Xlagnesiuin nitrate added before charring 3Iagnr&iiiri nitrate :rddcd aftcr cliarri,no 3IarnPsiiiiii nitr-otv ndde(l h i o r e ciiaiiini.
2.0 2 , .i 4.0 .i .0
0.0 .
~
2
3
4
32
77
91 97
_ _ _ _ _ ~ _ ~ ~ ~
~
5
I
7
8
9
10
MICROGRAMS ARSENIC
Figure 2. Standardization Curves Showing Per Cent Transmittance at Different Levels of Arsenic ’4.
B.
865 m p wave-length light soiirrc 840 m p wave-length light -ourre
that a good stantlard curve can be obtained using wxve lrngths near 840 mp, but that the rradings are not as sensitive :IS :It this wave length and that the wave lcmgth used mupt be kept constant. \lETHOD FOR I’HEI’AHATIO3 O F SAMPLES FOH ARSENIC DISTI1,LATION
ride or magnesium nitrate was added before the eggs m-ere charred rather than after charring, a much better recovery (67%) occurred. Magnesium nitrate appeared to give a better ash than the chloride, and further work was done with the nit,rate (Table I). Best recoveries of added arsenic were obtained when 6.0 ml. of saturated magnesium nitrate solution ivere added to 20 ml. of liquid egg before the egg was charred over n 3Ieker burncr as described. The arsenic content of several eggs from hens fed different levels of arsanilic acid or ,~-nitro-~-hydrosy~~lieri~~l:trsonic wids mas determined by the magnesium nitrate ashiny procedure described above. The values obtained were compared to those of similar eggs determined by the Gutzeit method. In all caws. esccpt for the eggs from the hens of group 1, Iiighcr vulues for arsenic were obtained by the ashing procedure than by the Gutzcit method (Table 11). Difficulty was esperienced by t,he analyst in obtaining what he considered reliable values for arsenic in eggs by the Gutzeit method. Group 3 hens received three times as much arsmilic acid as group 2 hens. Group 1 hens received no added arsenic, and group 4 hcns rrceived the Rriitro-4-hydrosyphenylarsonic acid. Table I11 presents data obtained with eggs from liens fed different levels of arsanilic acid, and the data are presented to show the reproducibility of results. Three or four eggs from each group of liens were composited and mised well before using. The first three values for each group were obtained from analyses made at the same time. The nest two a-ere duplicates in another set of analyses. An additional determination was made in duplicate for the last group of eggs. For the most part, the five values for each group of eggs agree well. Some of the valuos xere low, probably hecause of some loss of arsenic in some of the earlier ashings when the magnesium nitrate solution was not well mised in the eggs. These are placed in parentheses in the table, and they were not used in calculating the average va1uc.s given in Table 111. Reasonably accurate and reproducible results can be obtained by the method described herein, where the large 20-gram samples of eggs are used and wherr cnch samplc is analyzed in triplicate. The magnesium nitrate ashing method was also used for the determination of arsenic in other biological materials. Sinetynine to 101% recovery of 10y of arsenic added as a solution of arsenic trioside or arsanilic acid to fresh (*hicken liver, and 94 to 97% recovery of j y of arsenic added as a solution of nrsrnic trioxitlr t o dried pig liver were obtained.
Table 11. Comparison of Magnesium Yitrate ishing \lethod with Standard Procedure for Determining Arsenic in Eggs
l’oor rwovery of : i r s c k trioside or :trranilic~ :wid (5 to 10 inicrograms) added to liquid rggs w:w obtained by t h c short hydrochloric acid digestion prowtlure of Kingslry :ind Schaffert
1 2 3 4
(si. of t h r h :irsenic trioxide :~tltletlto eggs was .lpprosiniately rc~wvrredafter the mislure h:id been dissolved :inti the organic matter oxidized by wet, digrstion with nitric and sulfuric acids until :L clear solution I V : ~ohtainctl ( 1 ) . Better rccovery of added :irwnic rould no doul)t h v r heen obtnined after a little practice \\.it11 the method, l)ut the n-et-tligwtion procedure was long :md time-wnsuming, :tnd requirrtl Inrge amounts of nitric acid to oxidize c-omgletely a11 of t h r f:it in whole eggs, thus introducing :ii,wiiiv :is a contaminant : m l nocesaitating tho usr of a siniilarly t rc:itrtl hl:ink with each samplv. I3andrnic~and Schnihlr (Sj tlcsrribed a method for ashing caggs with magnesium rhloritlr. The method was used by Bandenier, D:iviclson, and Svhaihle ( 2 ) to determine the iron content, o f rgg whitw and yo1k.i. When c’ggs were ashed as described for the tlrterniination of iron in egg white, only 20% of the added nrsenir WLP rcv.vvcwd (TiiI)I(~I ) . When either magnesium chlo-
.irsenic in ‘&‘hole Eggs, y As/Grani Standard method 3lngnesinni nitrate met?;;;rl 0.005 iiig IIeiis
Group l a , Group 2 b , Group 3 C , Tinslie y As/Gram y As/Gram y As/C;iain Li\.er 0.07 0.86 1 33 Breast muscle 0.02 0.08 0 13 Leg muscle 0.06 0.11 0.17 Skin 0.01 0.12 0.21 0 Group 1 hens were fed no arsenic containing compounds. b Group 2 hens were given 90 grams of arsanilic acid per ton of feed C Group 3 hens were given 180 grams of arsanilic acid per ton of feed
LITERATURE C I T E D
(1)
h S 0 C .
Offic. Agr. Chemists, “Official Methods of iinalysis,”
7thed., 1950. ( 2 ) Bandemer, S. L., Davidsoii, .J. -I., and Schaible, P. J., Poultry Sci., 23,437 (1944). (3) Bandemer, S. L., and Schaihle, P. J., ISD. ENG.CHEY.,-4S.41..
Arsenic was determined in livers, breast muscle, leg muscle, and skin of hens and in liver, kidney, and leg muscle tissue of baby pigs. Some of these animals had received arsanilic acid. The fresh livers were homogenized in a lt7aring Blendor without the addition of water, the muscle tissue and skin were ground in a meat grinder, and the dried pig tissues were finely ground. Typical results obtained with fresh chicken tissues are presented in Table IV.
ED.,16,417 (1944). (4) Boltz, D. F., and Mellon, 11. G., ANAL.CHEM.,19, 873 (1947). (5) Case, 0. P., Ibid., 20, 902 (1948). (6) Chaney, A. L., and ,\Iagnuson, H . J., IND.ENG.CHEM.,ANAL. ED.,12,691 (1940). (7) Hubbard, D. &I., Ibid., 13, 915 (1941). (8) Kingsley, G. R., and Schaffert, R. R., A x . 4 ~ .CHEM.,23, 914 (1951). (9) AIagnuson, H. J., and Watson, E. R., IND. EKG.CHEY.,Ah-.41.. ED.,16,33b (1944). (IO) Maren, T. H . , I b i d . , 18, 521 (1946). (11) Morris, H. J., and Caivery. FI. O., Ibid., 9, 447 (1937). (12) Satl. Bur. Standards, Letter C‘irc. 929 (1948). (13) Sultaaberger, J. -4.,ISD. EXG. CHEM.,ANAL.ED., 15, 408 (1943).
4CKNOWLEDGMEhT
The eggs used for the data presented in Table I1 were fuinished by D. Tr. Frost of the Abbott Laboratories and were obtained by him from W. W. Cravens and M. L. Sunde of the University of Wisconsin Poultry Department. The arsenic determinations on eggs from the same source by the standard Gutzeit method (1) were made by J. B. Thompson at the Trace Metals Research Laboratories in Chicago, and the data were kindly furnished by D. 5’. Frost and are used with his permission.
R E C E I V E D for review March 22. 1953. Accepted November 5 , 19.53. Published with the approval of the Director of the Michigan Agricultural Experit iiient Station as Journal drticle No. 1471. Partially supported by a granfioiii the Ahhott Lahoratories, S o r t h Chicago, Ill.
Flame Photometric Determination of Sodium and Potassium In Zinc Cadmium Sulfide Phosphors SAMUEL B. DEAL T u b e Department, Radio Corp. o f America, Lancarter, Pa.
of cathode ray tubes, the presence of a I-small manufacture amount of an alkali silicate on the phosphor layer of T THE
the fluorescent screen is necessary from many standpoint.. Because no satisfactory chemical method x a s available foi the determination of small amount. of sodium and potassiuni in zinc cadmium sulfide phosphors, the flame photometer was used in the determination of these elements. The flame photometric method constitutes an accurate, rapid, and efficient procedure for the determination of concentrations of the ordw of magnitude of 0.0001 to 0.001% for sodium and 0.001 to 0.02% for potassium. Although separate samples were used for sodium and potassium in the analyses described here, the use of the same sample for both elements can be readily adopted. STANDARD S O L U T I O h S
Calibration curves for the determination of sodium and potassium with the flame photometer were obtained by analyzing a series of standard solutions containing known concentrations of the two elements. The standard solutions were prepared by adding known amounts of sodium and potassium salts to solutions containing sodium- and potassium-free zinc sulfate and cadmium sulfate in amounts equivalent to the weight of the samples to be tested. This method of preparing the standard solutions was used to reduce the effect of interfering ions. Berry et al. (1)and Parks et al. ( 2 )have shown that the presence of salts, ions, and acids in the solutions may lead to erroneous results unless all constituents are present to approximately the same extent
in both the standard and the sample solutions. An alteriiatiw procedure t o the method described here is to incorporate an internal standard in all solutions. DBJ The range of concentrations of sodium and potassium used in the standard solutions was such that it would include the range of concentrations found in the samples to be tested. The amounts of zinc sulfate and cadmium sulfate used were sufficient t o approximate the final composition of the sample solutions. Sepa-
Table I. Potassium Calibration Curve D a t a Potassium Content of Standard Solutions, P P. h.I 5 5 5
.
.
Meter Reading 100 100 100
4
80 81 80
3 3 3
52 50 50
2 2 2
26 27 27
4
4
12 13 13
