NOTES ON ANALYTICAL PROCEDURES Modification of the Arsenic M e t h o d of Magnuson a n d Watson Department of Parasitology, School
THOMAS H. MARE" of H y g i e n e and Public Health, Johns Hopkins University, Baltimore, Md.
T
HE method of hlagnuson and ,Katsoli ( 1 ) for the microde-
t'erniination of arsenic in biological materials was selected for use in :I program concerned with the pharmacology of arsenical drugs and their application to t'he chemotherapy of certain t,ropicul diseases. Preliminary tests indicated that recoveri.es viere within 5 to 10% of t,heoretical in the 1- to 10-microgram range, and the method vias therefore c ~ fthe required accuracy. The choice of this method over other> which also utilize the molybdenum blue reaction was based chiefly on the rapidity Kith whicli sarnples can be run t'hrough. As the authors state, 20 to 30 determinations can be done earl1 day with no difficult'y. A simplp microstill, commercially availnhlr,. is the only ypcial a,pparttus required. In this laboratory positive blank& and aberrant high values m-erc ocea-ionally encountered in handling this technique. The present paper describes an invest,igation of the cause of these falsc rradings, and indicates how they can be eliminated by :i mndificatiun of one of the reagents.
The following steps are carried out ill the determination of arsenic in blood, excrct.a, and organs. Details are described in t h r original paper ( 1 ) . The organic material is destroyed in a wet digestion using nitric a n d sulfuric acid. Perchloric acid may also be used a t the end of r l i c , process. Following digestion the arsenic is in pentavalent form in Yulfuric acid. This is transferred to the microstill, potassium hromide is added, and arsenic, probably in the form of the pmtabromide, is distilled together with a. small but significant Itmount of hydrobromic acid. The distillate is treated with ammonium molybdate, to form w. hrteropoly compound with arsenic as the central atom in the wniplex ( 2 ) . The heteropole is reduced with hydrazine sulfate to form molybIlenum blue, which gives a color with a sharp absorption maximum a t 840 m@. In the final solution, 1 part of arsenic in 25,000,000 may be readily detected and measured, using a Beckman ilxintrophotometer and a light path of 50 mm.
MODIFIED PROCEDURE
In order to avoid the handling of an extra holution, the cxtrr. milliequivalent of hydrochloric acid is added along with the ammonium molybdate solution. To two volumes of molybdate color reagent, made u p according t o tho directions of hIagnusor. and Watson, add one volume of S hydrochloric acid. I n the color development procedure, add 3 nil. of this combined solution instead of 2 ml. of molybdate solution as called for in the earlier paper. All other solutions and directioiis are unchanged.
Woods and Mellon (2) found that in the analogous case of phosphorus thc hydrochloric acid--molybdat,e ratio vias important. If this ratio w:iq too low, the reaction corresponding to the fourth step n-ould involve reduction of ammonium molybdate itself, t o form molybdenum blue even in the absence of the heteropole. Thus, in aiiy method of this type it is necessary to have the acid sufficiently
SUMMARY
A study has been made of the hIagnuson and Watson molybda-
Present address, Pharmacology Department, School of Medicine, Johns Hopkins Cniversity, Baltimore, Md. 1
num blue method for arsenic in biological materials, which offers certain advantages over others described in the literature. I t waF found that in their distillation process, insufficient acid was carried over, and the result was occasional high values in tlie presence of arsenic and posit,ive readings in the absence of arsenic and the other possible interfering elements, silicon and p h o ~ phorus. This was ascribed t o reduction of simple molybdate ion in insufficiently acid solution, and has been eliminated by addition of hydrochloric acid to the distillate.
I.
False Positive Arsenic Readin s D u e to Low Halogen A c i d Concentration in Magnuson-batson Procedure (All reagents tested and free of As, Si, a n d P. Readings from standard As curve, using Coleman Spectrophotometer a t 790 ma.) UndiDistilled Milliequivalents blilliequivalents nf HC1 used Reading a s As,y of H B r found Reading a s As, y 0 14.5 1.8 1.6 1.0 1.5,1.7,2.7 2.0,2.0 1.0,2.0 1.5 1.4.0.6 2.1 1.3 2.0 0.0,1.0,0.6 3.1,3.1 0 2.5 0.0,o 3.5 0 3.0 0,o. 0 4 . 0 (5-minute 0 distillation) 3.5 0,0 , o
Table
ACKNOWLEDGMENT
The author wishes t o thank Mildred Salchunas for her assisrance in this work. LITERATURE CITED
hlagnuson, H. J., and Watson, E., IXD.ENG.CHEM.,AN.iL. ED. 16,339 (1944). (2) Woods, J. T., with Mellon, M.G., Ibid., 13, 760 (1941). (1)
4.0
5.0
high so that only the heteropole, tvliivh contains the eleiiieni sought, is reduced. Magnuson and iT:itson ( I ) depended on the ibed 4-minute period for hydrobromic acid distilled in the p o 4.0 millicquivalents ol this purpose. They state ( a ) t,hat hydrobromic acid a,re distilled in this timc and ( h ) that as low as 1.0 milliequivalent is permissible. In thi,ir experiments, as &-ell a s the author's, hydrobromic and hydrochloric arid !yere used interchangeably in the color development steps. Ten successive distillations were r u n following tlie MagnuauriIJ-atson procedurti esactly. The distillat(+ n-ere titrated wit1 0.1 S sodium hydroxide using bromothymol blue as indicator Hydrobromic acid in the distillates \!-a< betn-een 1.3 and 3.6 m.e This range is 101%-erthan that given by Magnuson and \Vats01 (u, above). The difference is significant, since the experiment recorded in Table I show that a positive blank occurs regularly i less than 2 m.e. of acid is present. Thus, the permissible lower limit of acidit'y in the original paper (b, above) was also subject t o revision in the author's hands. Table I indicates that over 2.4 me. of halogen acid must be present t o avoid formation of molybdenum blue in t,lie absence of arsenic. I n view of these findings it has been possible to eliminate the occasional high values and false positives by the addition o! 1 m.e. of hydrochloric acid to the distillate. Since the lowest titer ever found for a distillate was 1.3 m.e., thic, increment should always be sufficient to bring the total amount of halogen acid t o the range where reduction of the simple molybdate and the resulting high values and false positives are completely eliminated Approximately 500 determinations have heen carried out with this modification, and no abPi,rnnt ri-iiltq of thr type origiritlly been observed. The over-all procedure has beer? ctory with regard to accuracy, speed, a.nd simplicity
WORKdone under a contract recommended b y t h e Committee on Medica Research, between t h e Office of Scientific Research a n d Development an.? t h e Johns Hopkins University.
0, 0.0
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