JULY 1947
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mercury was considered complete. If a cloudiness resulted digestion was continued until the supernatant liquid TTas clear. The cold finger was washed down with water and removed. The mercury was filtered through a KO. 2 Gooch crucible and washed by decantation 2 or 3 times with 10-ml. portions of water and then with one 5-ml. portion of 2 N sulfuric acid. The washing was continued with water until the filtrate gave no indication of chloride ion with silver nitrate solution. Two milliliters of concentrated nitric acid were added to the test tube and the resulting mercury solution was transferred to a 125-ml. Erlenmeyer flask. Using a small stirring rod the asbestos was transferred from the crucible to the Erlenmeyer flask. The crucible was placed over the opening of the flask, and 2 ml. of hot concentrated nitric acid were poured around the inside of the crucible in such a way as to dissolve any finely divided mercury which might he clinging to the wall and permit the acid to drain directly into the flask. The solution was diluted to about 30 ml., potassium permanganate solution added drop by drop in slight excess and the excess destroyed with a drop of ferrous sulfate solution. After the solution had been cooled to about 15’ C., 0.4 ml. of ferric alum indicator was added and the solution was titrated with standard potassium thiocyanate solution.
crease the effectiveness of the cold finger and would also neceesitate the use of a rather cumbersome oil bath. I n some instances when water was added to the clear supernatant liquid during digestion t o determine whether or not reduction was complete, a two-phase liquid system resulted on cooling and was a t first mistaken for finely divided mercury. However, upon reheating, the phases merged into one, indicating that the cloudiness was due to the organic reduction product which came out of solution a t the lower temperature. Since good results were obtained for the organic compounds analyzed, and since the mercury was attached directly to the benzene ring in most cases, it is believed that this procedure is general in application. LITERATURE CITED
Anfinsen, H., unpublished work, University of Pennsylvania, 1938. Danford, N. S., McNabb, W. M., and Wagner, E. C., unpublished work, University of Pennsylvania, 1939. Fenimore, E. P., and Wagner, E. C., J. A m . Chem. Sac., 53,2469 (1931). Low, “Technical Methods of Ore Analysis,” 8th ed., p . 181,New York, John Wiley & Sons, 1919. Rauscher, W. H., IND.EXG. CHEW, ANAL. ED.,10, 331-3 (1938). Sloviter, H.A.,McNabb, W. M.,and Wagner, E. C., Ibid., 13, 890-3 (1941). Staple, E., unpublished work, University of Pennsylvania, 1940. Willard, H. H . , and Boldyreff, A. W.,-J. Am. Chem. Sac., 52. 569-74 (1930).
The results obtained in the analysis of eight organic compounds are shown in Table 111. An oil bath was necessary for the digestion because severe bumping resulted when the test tube containing the mercury was heated directly with a microburn.er. Boiling stones were ineffective. Paraffin oil contained in a 150-ml. beaker served as a convenient bath for the digestion. The temperature of the bath was safely maintained between 115O and 120’ C. without danger of-bumping in the test tube. When water was substituted for the oil low results were obtained in the analysis. Samples larger than those designated are not recommended because too much water must be added t o the reduction mixture to ensure complete precipitation of the mercury. This would de-
ABSTRACTEDfrom the dissertation presented by Joseph N. Bsrtlett t o t h e faculty of the Graduate School of the University of Pennsylvania in p a r t i d fulfillment of the requirements for the degree of doctor of philosophy, June 1946.
Colorimetric Microdetermination of Antimony with Rhodamine B THO3IAS H. MAREN’ Department of Parasitology, School of Hygiene and Public Health, T h e Johns Hopkins University, Baltimore, M d . Modifications of a method for the colorimetric determination of antimony in biological material are reported, a second technique is presented which has certain advantages over the original, and the reactions involved are discussed.
IN
A preliminary report from this laboratory ( I S ) , a method for the colorimetric determination of antimony in biological material mas described. The purposes of the present paper are to present and modify that procedure in the light of more extensive experience, to report a second technique which has certain advantages over the original, and to discuss the reactions involved in these methods. The impetus for the research described in these publications came from the use of antimony compounds in the treatment of several tropical diseases, which were of particular importance to this country during the war. Antimony therapy has been fairly n-idespread in the tropics since about 1910, but the pharmacological data to govern its clinical use have been inadequate, largely because of the lack of a suitable analytical tool. The colorimetric methods presented here have proved useful in laboratory and clinical work in this direction. I t has been possible t o study the distribution of antimony in animal organs (18) and to follow 1 Present address, Department of Pharmacology and E xperimentsl Therapeutics, School of Medicine, Johns Hopkins University, Baltimore, Md.
-
blood levels and excretion rates in experimental animals and in patients undergoing therapy (19). Up to the present decade, chemical methods of determining the distribution of antimony in biological material were carried out chiefly by estimation of antimony sulfide or by adaptations of the Gutzeit procedure. Goodwin and Page (8) have summarized this early work, and have developed a polarographic procedure which was suitable for fairly small (10 micrograms) amounts of antimony in plasma and urine. Since the completion of the colorimetric methods reported here, McChesney (12) has described the application of the iodoantimonite reaction to samples of biological material containing a minimum of 10 micrograms of antimony. From known blood levels of other heavy metals it seemed reasonable t o assume that antimony blood levels during the course of therapy might well fall below 1 microgram per gram. The data obtained with the method described here and the studies by Brady et al. ( 1 ) and Cowie et al. (3) using radioactive antimony have amply confirmed this assumption. The best opportunity t o develop a simple method effective a t
VOLUME
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tion, at the same temperature at which nitric acid or peroxide .4 B C D was used. I t therefore appeared probable that volatiliDigestion zation w ~ t snot responsible for the losses encountered. I I This led to a study of the r-1none no,ne n o r Reduction nope none none Sq, valency state of antimony folI lowing sulfuric and nitric dinone rLi+none rLi+ gestion. Ce'Y + none none none The reaction with S b recovered rhodamine B served as a good 4 12 0 20 1 4 10 10 10 1 10 10 Micrograms 20 60 0 100 10 40 10 100 100 100 100 100 % indicator, since only the pentaProbable valency s t a t e after digestion step valent antimony gives the color 20 0 10 100 Sb", % reaction. Under the proper 100 30 40 0 0 40 60 0 conditions of acidity (20),hoM 40% of S b was A11 S b was recov- Although a large amount 911 S b was recovered not recovered ered a n d vaof "SbIv" was formed and valency was S b l ever, the trivalent metal may lency was SbIII a n d did not (as i n b) i n digest, i n throughout, after perbe completely oxidized by ceric after digest. correspond to chloric oxidation this case reduction of HrSOa alone is SblII or SbT'. a portion of digest sulfate to the pentavalent stat?. This "lost" prior to Ce treatment not a practical fraction way of dehas led t o complete reWebster's benzene extraction stroying orcovery in t h a t portion been called method, vhich includes this ganic material Sb'" oxidation system, was used i n these studies, which are sum. marized in Table I and discussed in the following paragraphs. In these experiments antithis level seemed to lie within the field of colorimetric organic remony sulfate was used as the standard. On undigested samples agents. this reagent (Sb"') gave no color if the ceric sulfate step aas Eegriwe (6) discovered that xanthone dyes formed a colored omitted, and yielded quantitative and reproducible results in the compound with SbV which was somewhat different, in its light prescribed Webster technique using this oxidizing agent. absorption, from the dye itself. Based on this reaction Fredrick (6) published the first paper on the quantitative colorimetric estiIt will be seen from Table I, column il, that only a small poimation of antimony with rhodamine B. This procedure is intion of the antimony, in this case 20%, is oxidized to the pentavolved, and as a result "about 47, of all analyses" failed in Fredvalent state by the sulfuric-nitric mixture. This was a typical rick's hands. Many interfering substances were encountered, and experiment, and although there n a s wide variation in this reacthere was no consideration of the preparation of the sample, a problem of primary importance in the analyses of biological mation, complete oxidation to the pentavalent state was very rare. terial. Kevertheless, this work indicated that rhodamine B was a This is in marked contrast to the behavior of arsenic, nhich is highly sensitive reagent for antimony. By making suitable volcompletely converted to AsV under similar conditions. When ceume and concentration adjustments in this technique, an absolute ric sulfate was added to the antimony residue, complete converlimit of 1 microgram metal in 10 nil. of final solution could be detected even with a simple colorimeter. sion to the pentavalent state is still not achieved; in the experiWebster (81)provided a considerable advance over Fredrick's ment indicated in Table I, A, this brought the recovery up to original method by introducing benzene to extract the metal-dye only 60%, and the remainder was lost. Table I, B, shows that in complex after its formation in the aqueous solution. This shortthe absence of an active oxidizing agent, such as nitric acid, the ened the procedure and added greatly to its reliability. Using this system, Webster and Fairhall (22) recently developed a proantimony remains in the trivalent state, and that under these cedure for the direct microdetermination of stibine gas. I n none conditions there is no loss when later oxidized with ceric sulfate. of this work, however, was the digestion problem considThis had also been shown by Hallman (9). Unfortunately, thi5 ered, or the application t o biological material studied. Provided cannot be applied practically, since sulfuric acid alone is satisfarwith the background of Fredrick's (6) paper and Webster's early work (RI), the problem was to develop a rapid and accurate toly only for the digestion of centigram quantities of tissue. method for the determination of antimony in all types of organic The data of Table I, columns -2 and B, suggested that the u5e material. of nitric acid in the wet-ash procedure n a i somehow responsible for the apparent losses encountered. Conrad ( 2 ) had sholln that DEVELOPMENT OF GEVERAL METHODS FOR BIOLOGICAL nitric acid oxidation of antimony vieldb a mixture of three oxides, SAMPLES Sb203,Sb204,and Sb?O,. In early 1%ork the present author had asDigestion and Preparation of Samples. Both in this laboratory sumed that even under such conditions the ceric sulfate used in thts and elsewhere (9) digestions of organic material containing anticolor development reaction would oxidize an intermediate form as mony were attempted by conventional wet-ash methods using Sb204to the reactive pentavalent state. Experience such as that nitric and sulfuric acids, which are in general use for the deterrecorded in column A of Table I suggests strongly that this is not mination of other metals such a6 aisenic and mercury (14, 15). the case, since there is an unreactive portion of antimony ever1 I t was possible to check these digestions either by Fredrick's after the ceric sulfate treatment. This unreactive portion is piooriginal method or by Webster's modification, using benzene visionally called Sb2O4,after Conrad ( 2 ) and is referred to in the. extraction. Both techniques called for a high concentration of table as Sb". S o investigation of the nature of the binding in thi. acid in the color-development stage, so that the completed digest molecule has been attempted, and these classifications have bern was suitable for direct application of these colorimetric reactions. retained for the sake of convenience. The tetroxide is clearlg a HonWer, following a nitric-sulfuric or peroxide-sulfuric digest, chemical entity with a reproducible empirical formula, corr: recovery of antimony was highly erratic, ranging from an apparsponding to that given here, since it has been used (IO)sucrtwent ccmplk'te loss of antimony to complete recovery. These fully for the gravimetric determination of antimony. losses xiyere a&ribed by earlier workers (11) to volatilization andSince direct oxidation of the antimony by the sulfuric-nitl1(. the elusive nature of the losses made this an obvious explanation. method had failed to convert the metal completely to the desircd Yet arsenic, which is generally more volatile, could be digested in pentavalent form, reduction was attempted prior to the ceric SUIthe very same way without loss. I t was also observed that small fate step. h critical experiment of this type is recorded in column amounts of organic material containing antimony could be C of Table I. Sodium sulfite was added to half of a sulfuric-nldigested without 10s if sulfuric acid alone was used in the digesTable I.
Valency State of A n t i m o n y a f t e r Digestion of Sb"'
r-
+1
-
LA+
JULY
1947
489
"7 13C
/
/
/'.,
-- -
i3en;jene-
\
Et he T
concentration of anions had a profound effect on the reactions. After completion of the author's work Rebster and Fairhall (22) published additional data on these reactions. 1. I t was found that conditions could be chosen so that 5 ml. of sulfuric acid, the residue from the digest, could be used directly in the color determination. These conditions are discussed in the following paragraphs. 2. Fredrick ( 6 ) had found that a high concentration of chloride ion was necessary and used lithium chloride for this purpose. Lithium was found to be an extraneous factor and hydrochloric acid was used. In the absence of hpdrochloric acid color values are entirely negative, and increase to a maximum when 5 ml. of 6 AV hydrochloric acid .are added. This was found generally true no matter what type of variation was made in later steps. 3. Webster's ( 2 1 ) use of phosphoric acid has been retained. I t serves the useful function of reducing the interference of iron and possibly that of other metals that form stable complexes with Pod. The amount selected here was the maximum that did not inhibit color development. This amount will cause low results, however, if the solution is allowed to stand after its addition. Following the phosphoric acid step, therefore, the procedure should be completed as rapidly as possible, and the extraction should be done within 5 minutes. 4. The amount of rhodamine B selected is suitable for a t least 300 micrograms of antimony. If more is encountered and the aqueous phase is entirely bleached during the extraction step, this indicates that more dye is needed, and it may be added directly. The final volume of dye solution was selected to give the proper concentration of reagents for quantitative recovery, using 10 ml. of benzene in the extraction. This volume of benzene is also suitable up to about 300 micrograms of antimony. If the dye solution is bleached, as described above, and more is added to ensure excess of the rhodamine B, it is also generally necessary to perform a second evtraction with 10 ml. of benzene, and this should be repeated until the solvent layer is colorless. Figure 1 shows the absorption peak of the antimony-rhodamine B complex in benzene to be a t 565 millimicrons. Figure 2 shows antimony concentration as a function of density readings on the KlettSummerson photoelectric colorimeter.
30u
.I 0
420
420
440
460
480
500
S2C .
540
560
680
$oO
k0
&O
Wave Length, M r
Figure 1. .ibsorption of SbV-Rhodamine in Benzene and Isopropyl Ether Concentration, 1 microgram of Sh per m l . of solvent Coleman No. 11 spectrophotometer with 50-mm. cells
tric digest, with subsequent heating to fumes of sulfur trioxide. Upon treatment of half of this residue with the usual reagents, including ceric sulfate, antimony was completely recovered. .hi unreduced quarter of t'he digest, similarly treated, yielded but 40y0 of the theoretical. Sodium sulfite does not reduce the amount of Sbv that is 'formed in the digestion, since this portion may be recovered without the use of ceric sulfate. Under these conditions sulfite is an indifferent reducing agent for pentavalent antimony, but this is only of incidental interest, since Sb" is the detectable form in the color reaction. However, it was significant that an unreactive portion had been entirely reduced, and that this form of antimony, although highly resistant t,o oxidation, is very susceptible to reduction. Lundell ( I O ) cited these as properties of the tetroxide, SbpOa,and Conrad (8)isolated this suhstance from a nitric acid oxidation process. Because of its unusual resistance to oxidation, neither nitric acid nor ceric sulfate had been able to bring antimony tetroxide to the desired pentavalent state. Conversely, the susceptibility of antimony tetroxide to reduction made it possible to use sulfur dioxide to convert it to Sb"', which in t,urn could be stoichiometrically oxidized by ceric sulfate to yield Sb'. With the adoption of the use of sodiuni sulfite after the digestion stage, quantitative recoveries were uniformly obtained. This method is described in a preliminary paper ( I S ) . I n the course of work involving the digestion of 10- to 15-gram samples of whole blood and tissue, it became convenient to add perchloric acid in the latter part of the digestion to hasten the process (13). This had the effect recorded in Table I, column D. In this experiment antimony was quantitatively oxidized in the digestion to the pentavalent state, a condition that was inipossihle with nitric acid alone. Since rhodamine B gives the required reaction lvith St)", the use of perchloric acid offered a means of eliminating sulfite reduction and consequent oxidation Tvith Ce" as originally described ( I S ) . This suggestion \vas successfully followed by Freedman (?) on several hundred known samples, and the procedures described below dispense with both these reagents. In other respects the present description of the benzene method (-4, belon-) is the same as that previously published ( I S ) . Reactions Used in Color Development. A . BEXZESE UETHOD.B number of factors were involved in the development of a scries of reactions to yield a reproducible color complex of mtimony and rhodamine B. These were investigated by Fredrick ( 6 ) and Webster (ZI),but the presence of a comparatively large amount, of sulfuric acid for digestion made a new study necessary. As in many colorimetric methods, the character and
IM
::
//
&n:ene Met hod Ether Method---
-
/
5 . It vias possible to digest 50 mi. of urine, 20 ml. of plasma, or 10 grams of nonferrous tissue, without encountering a positive reading on knorm blanks. Larger amounts could probably be used, but these are the approximate limits of convenience with the digestion technique described. However, if more than 2 ml. of whole blood or other iron-containing tissue was used, a positive blank was found. Iron could be present up t o about 1 mg. without interfering with the determination of 1 microgram of antimony, but above this quantity of iron, either an inorganic salt
V O L U M E 19, NO. 7
490
or as hemoglobin in tissues, a positive blank was encountered (Tables I1 and 111). Since 10 ml. of blood contain 2 to 3 mg. of iron, and since this volume of blood must be used to detect levels of 0.1 to 0.2 microgram of antimony per gram which are observed after administration of trivalent antimony, it was necessary to eliminate this source of error. It could be greatly reduced, and was a predictable value if the temperature was kept constant at 20” to 23” during extraction. This value, as indicated in Table 11, is 0.8 microgram for 10 ml. of blood. The normal reagent or “extraction” blank is 0.4 microgram, so that the iron error is significant only at the lowest range of observation. Severtheless, a means of completely eliminating it was sought. B. ISOPROPYL ETHERMETHOD.A possible means of separating iron from antimony was offered by the fact that Fel” is quantitatively extracted from 6 to 8 N hydrochloric acid by ether or isopropyl ether (4, l 7 ) , whereas Sb”’ is almost entirely retained in the aqueous phase (16). This was found true for the author’s experimental conditions, using the trivalent antimony sulfate standard and isopropyl ether. However, this procedure presents certain difficulties. I t would be necessary to introduce new reagents and a method of reducing all the digested antimony to the trivalent state. Sulfite reduced only the antimony tetroxide and not the pentavalent form. Traces of peroxides in the isopropyl ether could oxidize the trivalent antimony to SbV, and thus bring about a partial extraction and loss into the ether phase. Mylius and Huttner (16) had previously reported the solubility of Sbv in ethyl ether. The finding that Sbv was extractable with isopropyl ether led to the development of a new method for separating iron from antimony. Further investigation revealed that pentavalent antimony is completely extracted from 1.5 &- hydrochloric acid by isopropyl ether. At this acid concentration, over 907, of the iron remains in the aqueous phase. It was then found possible to develop the Sbv-rhodamine B color directly in the ether. Aliphatic ethers may not be used to extract the complex from acid solution in the way that benzene and certain other aromatic solvents are used, since in the case of ethers the partition coefficient favors the aqueous solution. Kevertheless, if the antimony is in the ether phase and dye is added to it, the colored complex will form in the solvent, whereas the rhodamine B itself d l remain in the water phase. The system Sb”-rhodamine B-isopropyl ether is not stable, and the color readings must be made within 30 minutes of the extraction. The colored complex is not truly soluble in the ether, and for this reason deviates slightly from Beer’s l a v (Figure 2). Table I1 shows that an absolute blank is obtained with 10 ml. of whole blood, which is not the case with the benzene method. Care must be taken that the digestion is actually complete before the color development is begun, particularly in the ether methbd, where traces of undigested material will cause opalescence in the solvent and the sample will be entirely spoiled. This may be avoided by heating the digest strongly to fumes of sulfur trioxide before i t is completed. CHEMICALS AND REAGENTS
Sulfuric acid, c.P., specific gravity 1.84. Nitric acid, c.P., specific gravity 1.42. Perokloric acid, c.P., 60%. Benzene, C.P. (for benzene method only). Isopropyl ether, c.P., Carbide and Carbon Chemicals Corp. (for ether method only). Capryl alcohol, C.P. Hydrochloric acid, C.P. (specific gravity 1.19). Make a 6 A’ solution by mixing with an equal volume of distilled water. Orthophosphoric acid, C.P. (specific gravity 1.71). Make a 3 A’ solution by diluting 70 ml. of concentrated acid to 1 liter with distilled water (for benzene method only). Rhodamine B. Eastman. Make 0.02% solution in distilled water. Antimony solution. Weigh out 0.1000 gram of C.P. antimony and add 25 ml. of concentrated sulfuric acid. Heat until the metal dissolves. Cool and dilute to 1 liter with distilled water. This solution contains 100 micrograms of trivalent antimony per
ml. The same standard has been used for 2 years, with no detectable change. I t is generally diluted to give working laboratory standards of 10 and 1 microgram per ml. APPARATUS
Erlenmeyer flask, 50 to 100 ml., or preferably, Stoll egg-counting flasks (A. H. Thomas Co.). Three-heat laboratory hot plates. Coleman No. 11 spectrophotometer, 11-100 cuvette carrier, matched round cuvettes of 19-mm. diameter, No. 11-101. The Klett-Summerson photoelectric colorimeter was also used, with round cuvettes of 13-mm. diameter, and a No. 54 green filter. Buret, pipets, small separatory funnels, small test tubes, or standard 15-ml. centrifuge tubes. PROCEDURE
The techniques for the benzene and ether methods are the same through addition of hydrochloric acid. Following t,his point directions are given separately. Digestion. Weigh out blood or tissue up to about 15 grams (50 ml. of urine or plasm6 may be used) into an Erlenmeyer or Stoll flask, and add 5 ml. of concentrated sulfuric acid and about 5 ml. of concentrated nitric acid. Add a few glass beads or Alundum chips and 1 drop of capryl alcohol. Allow the digestion to begin spontaneously; shake to aid solution. The digestion may be left to stand a t this or any later stage. Place the flask on the hot plate a t low heat, and after nitric acid fumes evolve and digestion is proceeding rapidly (1 to 2 hours) advance the heat to medium. If charring occurs remove from hot plate, allow to cool somewhat, and add 1 to 2 ml. of nitric acid. Shake for several seconds and return the flask to the hot plate. I t may be necessary to repeat this process several times. *JThenno further charring is evident, advance the heat to high. If iron is present the solution will be yellow when hot, but colorless (often with a white granular precipitate) !Then cool.
Table 11. R e c o v e r y o f A n t i m o n y f r o m 10 311. of Whole Blood
Sb Added Y
0 1 2 5 10 20
Benzene Recovered, average minus blank Recovered Y
0.8,0.8,0.8 1.8,1.8,1.4
3 0,3.0,2.5
5.9,5.5,4.8 11.2,11 0,10.6 20.5,20.2,20 2
Isopropyl Ether
Recovered Y
Y
(0) 0.9 2 0 4.7 9.8 19.5
0,o 0.8,0.8 2.0,1.8,1.8 4.8,4.2,4.2 10.8,10.8,9.2 22,21,21
Recovered, average /
0 0.8
1.8 4.4 10.3 21.3
When the cooled solution is water-white or only slightly yellow and no further charring occurs, add 2 drops of perchloric acid and heat strongly to fumes of sulfur trioxide on maximum hot-plate temperature. Occasionally this will cause charring or yellowing, and this indicates that more nitric acid is needed. I n such a case, perchloric acid treatment must follow. Generally, this perchloric treatment serves to finish the digestion, and the cooled solution will be water-white. After perchloric treatment add 3 ml. of water and heat until sulfur dioxide fumes evolve. The time necessary to carry out a digestion varies greatly with the type and weight of sample used. The author’s procedure has been to run up to 50 samples a t one time. Fifteen milliliters of whole blood will take about 20 hours, and 15 ml. of plasma about 4 hours. Color Development. I t is advisable to carry out this operation in a cold water bath, since considerable heat is liberated and it is desirable to extract under 23 C. Bdd 5 ml. of 6 ,V hydrochloric acid. If a deep yellow color develops it is due to iron in excess of l mg., and if the amount of antimony expected is very small, the ether method (B) should be used. Otherwise a t this stage method A is suggested. METHOD.Add 8 ml. of 3 N phosphoric acid and A. BEXZENE 5 ml. of 0.02’%rhodamine B. Shake the flask and cool if necessary. Do not delay a t any point after the addition of phosphoric acid. Pour the solution into a separatory funnel, and add 10 ml. of benzene to the flask. Pour this into the separatory funnel and shake 150 times. Draw o f f the aqueous layer and collect the benzene phase (colored red if antimony is present) in a test tube or
JULY
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,
491
1947
small centrifuge tube. Let stand for several minut,es to allow any droplets of water to settle. Centrifugation is not necessary. Pour off 6 to 8 ml. into t,he cuvette for estimation of color. The solution is stable for a t least 4 hours, and can be left overnight at the risk of only minute change. Read at wave band 565 mp, or in the absence of a variable wave band, use a green filter. Figure 2 relates concentration of antimony to dial readings on the KlettSunimerson instrument, using the green filtrr. B. ETHERMETHOD. -4fter the addition of hydrochloric acid add 13 ml. of water, and pour the solution into a separatory funricl. .4dd 15 ml. of isopropyl ether to the flask, and pour this into the separatory funnel. Shake 100 times, and discard the lower aqueous layer, leaving the ether containing the antimony in the separatory funnel. AAdd5 ml. of rhodamine B solution, and shake 1.50 times. Discard the lower aqueous layer and collect the ethereal portion in a small test or centrifuge tube. Read immediately, a t 545 mM, or with a green filter on the Klet,t-Summerson. Figure 2 relates concentration of antimony to optical readings on this instrumrnt. With either method, the extract may be diluted with the appropriate solvent if the color is too intense to fall within the range of the reference curve. I n such a case it map also be necessary to perform a second extraction on the aqueous phase, using fresh solvent. The second portion is estimated in the usual way and its value added t>othat of the first extract. Standard Reference Curve. Using the dilute standards, add incrcrnental values from 0 t o 40 micrograms of ant,imony to 5 nil. of sulfuric acid. Treat, this with nitric and perchloric acids as above and continue with the procedure in the regular way.
an hour. The author has found it convenient to cairy siriipleu through t o the color stage in groups of five or six. Digebtion consumes most of the time involved. The maximum time required is for digestion of red cell material. .4 single operator was able to run 52 such samples (about 8 grams each) in 4 days. Feces and fat are also time-consuming, and it is inadvisable to u-e more than a fen- grams.
Table 111. Interference of Other RIetals w i t h 4ritinionyRhodamine B Reaction in Benzene -1. Lnterference with blank, no S b present Micrograms of Foreign hletal Present 100 1000 10,000 Colorimeter Reading, Micrograms of S b 1 6 0 1.0 0 0 0
0
0
0 0
0 6
0 n n 0 0 0 2 0 0 0 0 0. hrilulsion formed n i t h benzene. volorless.
B.
I n t e r f e r e i i i ~ Kith 2 y of S b hIicrograms of Foreign Metal Prerent 100 1000 10,000 Colorinieter Reading, 12Iicrograrris of Sb 2.4 2 8 4 0 2.0 2.4 1.0 2.4 1 8 2.5 2.4 2.0 " 0 1.3 0' 2.2 2.2 2.2 2.4 2.0 2.2 2.0 2.2 6.2 2.6 2.1 2.0 2.0 2.0 2.0 0" 2.3 2.0 2.4 When broken, snlreiit layer was
DISCUSSION
I n the construction of the standard reference curvcs for both methods (Figure 2) it was found that optical density readings n-ere reproducible within 20% up to 2 micrograms, and within 107; for higher values. The same recoveries were observed when known amounts of antimony were added to urine and plasma. I n all these cases an absolute blank is obtained in the absence of antimony-Le., optical density readings are no greater than that obtained when benzene or ether is shaken with water. Since the author has used the pure, unused solvent as the reference in all cases, the actual reading for the extracted solvent is a positive one, due to t,he slight solubility of water in the organic phase. This value for benzene would correspond to 0.4 microgram, and for ether to 0.2 microgram of antimony. These figures mark the loner absolute limit of the method, and are recorded as zero antimony. Table I1 s h o m recovery of antimony from 10 ml. of whole blood by both methods. A positive blank, due to iron, is obtained with the benzene technique, and this is eliminated in the ether method. The ether method is somewhat less accurate for individual readings, perhaps because the procedure involves two extractions and the dye has a low solubility in the solvent. The interference of other metals with the benzene method is recorded in Table 111. I n studying this factor, attention has been given bot,h to interference by formation of a positive blank (column A), and to the masking of a true antimony reading by a foreign element (column B). It is also evident that interferences are relative rather than absolute in nature, since they are observed a t certain definite concentrations of the elements in question. At a level of 100 micrograms none of the eleven elements interferes either with a blank or with the determination of 2 micrograms of antimony. At a level of 1000 micrograms, only arsenic interfereq with the blank, while arsenic, tin, and iron increase the 2-microgram reading. K i t h the next tenfold increment of metal, interference becomes more common, although the presence of even these very large amounts of bismuth, zinc, lead, mercury, molybdenum, cobalt, and probably copper is permissible. The procedure has been applied only to biological materials, but it should be possible to adapt it to the other analytical uses, particularly in view of its high specificity. The time required for the color development itself is small. On inorganic material it is possible to run ten drterminations in
Thebe methods have been used succebsfully in the clinical laboratory of a small hospital in the Kest Indier Ivith no ipecial preparation other than the addition of a hood: 385 determinatinns were carried out in a 3-week period. ACKNOWLEDGMENT
The authoi ~ i s h e bto thank G. F. Otto for iugge>tioii-,H. O., I N D .ENG.CHEM... ~ N . A L .ED.,
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e,,
(in press). (20) Rathsburg, H., Ber., 61,1663 (1928). 121) R'ebster, S. H., cited by Hallman in (9). ( 2 2 ) Webster, S. H.. arid Fairhall, L. T., J . I n d . Hug. 'Ibxicol.. 27, 183 (1945).
