494
VOLUME
ful workmanship during the development of the syringe microburet. LITERATURE CITED
Chaney, A. L., IND. ENG.CHEW,h . 4 ~ ED., . 10, 326 (1938). Clark, W. G., Levitan, Ii. I., Gleason, D. F., and Greenberg, G., J . B i d . Chem., 145, 85 (1942). (3) Dean, R. B., and Fetcher, E . S., Jr., Science, 96, 237 (1942). (4) Hadfield, I. H., J . Soc. Chem. Ind., 61, 45 (1942). . CHEM.,.&SAL. ED.,7. 180 (1935). (5) Krogh, 4.,1 s ~EXG.
(1) (2)
19,
NO. 7
( 6 ) Kiugh, .&.. and Keys, A. B., J . Chem. SOC.,1931, 2436. (7) Office of Publication Board, U. S. Department of Commerce, Report PB 5924 (1946). Sasaki, N., 2 . anorg. allgem. Chem., 137, 181 (1924). Scholander, P. F., Edwards, G. .1.,and Irving. L.. ,J. Biot. Chem., 148, 495 (1943). (lo) Tievan, J . ii.,Biochem. J . , 19, 1111 (1925). COXTRIBUTIOX 1107 from the Gates and Crellin 1,aboratories of Chemistry, California Institute of Technology. Based upon work done for the Office of Scientific Research a n d Development under Contract OEJIsr-325 with t h e California Institute of Technology.
Determination of Mercury in Biological Material F. L. KOZELKA. Uninersity of Wisconsin, Madison, Wis.
A simplified dithizone method for the determination of mercury in biological material is described. The uniqueness of this new method is the quantitative distillation of mercury from the Kjeldahl flask during and subsequent to the digestion without the loss of the metal. This procedure separates the mercury from all the nonvolatile salts of the other metals contained in the specimen. Washing t,he mercury di-
T
HE most dificult thing t o achieve in the detrrniiIiation of mercury is the complete destruction of the organic matter without t,heloss of the metal. The volatility and lack of stability of mercury salts at higher temperatures complicate the deromposition and preparation of the sample for analysis: hence suitable precautions must be taken t o prevent losses through volatilization. To minimize this loss, most of the available mtxthods suggest the use of potassium permanganate or potassium chloratta partially to destroy the organic material, with subsequent concentration of the mercury by precipitat,ion as a sulfide. Kinklei, (b)employed zinc as an entraining agent for mrrcury from nitric. acid extracts of vegetables. The precipitatr containing the mercury was then dissolved in nitric acid and the sinall quantity of' organic matter present was osidized with potassium permanganate. These procedures are time-consuming and an appreciable error may be introduced, particularly when microquantities of mercury are involved either by incomplete entrainnient of thP mercury or the intmdur.t ion of mme mercury with the rragenth employed. -4nother difficulty tsricwuntered in the existing nirbthoda is in the separation of mercury from copper, since both metals combine with dithizone under wsentially the same conditiorib. Winkler (5) employed sodium thiosulfate, while Laug and Nelson (4) used potassium bromide t o effect this separation. Both of these compounds complex the mercury and transfer it from the chloroform phase t o the aqueous phase without affecting the copper dithizonate. In either case the procedure requires several transfers and digestions which are time-consuming for routine purposes. Gettler and Lehman ( 2 ) adapted the potassium permanganate digestion to small quantities of urine and extracted the mercury from the digest lyith dithizone. The technique, however, is applicable only to specimens in which the final digest is small and the quantity of mercury relatively large. Hubbard (3) digested 50-ml. urine specimens with potassium permanganate but substituted di-p-naphthylthiocarbazone for dithizone since they exhibited the same general characteristics. Subsequently, Cholak and Hubbard ( I ) applied this technique t o tissues, employing a preliminary digestion with sulfuric and nitric acids followed by a final digestion with pot'assium permanpxnate. I n preliminary studies in this laboratory the writer observed that the loss of mercury from a digest was markedly increased with a n increase in the chloride ion. and suhsequent wnrk demon-
thizonate with 9 S ammonium hydroxide obviates the necessity of treating the dithizonate extract with potassium bromide or sodium thiosulfate and performing a second digestion and extraction,as required by the pretiously described methods. Consistent reco,eries with an error of less than 2 micrograms are obtainable, which appears to he adequate for toxirolopical or clinical purposes. strated that niercury can be distilled quantitatively from the'digePt by the addition of chlorides as sodium chloride, hydrochloric acid, or chlorine gas. The disadvantage in the use of sodium chloride is the excessive accumulation of sodium sulfate in the digest, too rapid liberation of chlorine, and excessive foaming. The use of hydrochloric acid results in a large volume of the final distillatc and a considerable amount of acid which mupt be neutralized hefore the merrury is extracted. The niajor portion of the mercury is apparently distilled as a mercury aninionium chloridr c,omples, because less than 20L; recoveries can he obtained in the absence of nitrogen. able over a wid? pH range, Snce the mcrcury dithizonate i it can ht, wparated from the excess dit,hizone and othcr contaminants (particularly the traces of copper on the glassware) by washing it in approximately $1 S ammonium hydroxide. The copper dithizonate dissociates in this medium n-ithoiit affecting the nwrc-iiry dithixonatr. *
.
REAGENTS . i N D APPARATUS
dulfuric acid, concentrated, C.P. quality. .Ammonium hydroxide, 9 N,redist,illed. This solution
Table I.
2.5 2.5 5.0
5.0 10.0 20.0 30.0 40.0 50.0
Liver
Kidney
pre-
Recoveries of Know-n Quantities of JIercury from 25 Grams of Tissue Mercury Added Ilzcroorams
Blood
ib
Mercury Recovered Mzcrograms 4.25 4.50 7.00 6.75 11.50 22.00 31.00 41.50 52.25
hrror h'zcrogravra 0.00 +0.25
+0.25 0.00 -0.25 1-0.25 -0.75 -0.25 +0.50
27.0 50.5 245 (1
+0.25
250.0 25 0 50 0 250.0
26.0 52 0 247.5
-0.75 + O 25 -4.25
25.0 50.0
Heagent blank contained .1 75
micrograms
-1.25
-6.75
of niercury
JULY
495
1947
pared by distilling concentrated ammonium hydroxide and then diluting it to the necessary concentration. Ammonium sulfate. Copper sulfate. Dithizone solution, approximately 5 mg. of dithizone per 100 ml. of redistilled carbon tetrachloride, purified before using. Filter paper, metal-free, 9-cm. Whatman No. 40 extracted with purified dithizone. The dithizone solution is changed periodically until metal-free, as determined by shaking a portion of it in dilute ammonium hydroxide. The excess dithizone is removed by successive washing with pure carbon tetrachloride. Chlorine gas. Sulfur dioxide. Thymol blue indicator. Apparatus as shown in Figure 1. The open end of the receiving flask is connected to an aspirator by means of a loose-fitting stopper to prevent the escape of the fumes into the laboratory. The receiving flask should be washed with nitric acid and rinsed with metal-free water before being used.
100 I
5000 6000 Wave Length, .ingstroms
4000
Figure 2.
7000
8000
Transmittance Curves
PROCEDURE
Tissues. Twenty-five grams of blood or other tissues are placed in the 800-ml. digest'ion flask. Glass beads and approximately 1.0 gram of copper sulfate and 15 to 20 grams of ammonium sulfate are added. The flask is then connected to the apparatus and concentrated sulfuric acid is added slowly through the funnel (50 ml. for blood and 75 ml. for other tissues). The digestion is started at a very slow rate and then slightly increased after the foaming has subsided. If the flame is too high considerable charring will occur and some carbon will be carried over with the distillate. Should this occur, the entire distillate should be returned to the flask and redigested after the digestion is practically complete. A certain amount of lipoidal material nil1 distill over: however, this can be filtered off without affecting theresults, After the digestion is completed, the chlorine gas tube (fitted with a small rubber collar) is fitted into the bottom of the funnel and the gas s 1o w 1y b u b b l e d through the digest. Simultaneously the digest is heated with a mediumsized flame. .4pproximately 20 minutes are required bo distill all t h e mercury. The chlorine tube is then removed and air is slowly aspirated through the apparatus and the distillate t o remove the excess chlorine and t o decompose the major portionof the hypochlorous acid by the sulfur dioxide generated from the decomposition of the sulfuric acid. This is accomplished by a p p l y i n g slight pressure on the stopper in the open end of the receiving flask. The distillate is then transferred t o a separaFigure 1. Apparatus tory funnel, filtered if necessary. It is A . 4 X 20 cm. test tubes B . 14/35 ground-glass joint advisable to bubble C. 34/65 ground-glass joint sulfur dioxide D . 1-em. tube through the speciE. 800-cc. Kjeldahl flask men before extracF. Water bath G: Glass beads tion for about one
0
5
10
15
20 25 30 35 40 Micrograms of 3Iercury
Figure 3.
45
50
55
60
AIercury Present
minute to ensure complete destruction of the hypochlorous acid and to prevent any decomposition of the dithizone. Urine. The desired volume of urine is measured into the digestion flask and acidified with approximately 5 ml. of concentrated sulfuric acid. Acidifying the urine minimizes the foaming Tvithout causing the loss of any mercury. Glass beads are added and the water is distilled off until the residue is reduced t o a volume of 30 to 50 ml. This distillate is discarded. The digestion of the residue and distillation of the mercury are then carried out as for tissues described above. Extraction of the Mercury. After the distillate has been transferred to a separatory funnel, sulfur dioxide is bubbled through the solution for about a minute. The solution is then neutralized to approximately pH 1 by the addition of concentrated ammonium hydroxide until the indicator (thymol blue) just changes from the red to yellow color. The solution is cooled under a tap, exactly 25 ml. of the purified dithizone are added, and the funnel is shaken for about a minute. The carbon tetrachloride containing the mercury dithizonate is then washed twice with approximately 50 ml. of 9 -V ammonium hydroxide to remove the excess dithizone and any copper dit'hizonate which may be present. In each of these transfers as much of the carbon tetrachloride is drained off as possible. A quantitative transfer is not necessary, since the mercury is uniformly distributed in the 25 ml. of carbon tetrachloride originally added and only 10 ml. are required for the colorimeter reading. The sample is then filtered through a dry metal-free filter paper into a colorimeter tube and a reading is taken. Mercury dithizonate is photosensitive and should be protected from bright light. I t is advisable to carry out the extraction in subdued daylight or artificial light. An Evelyn photoelectric colorimeter with a filter centered a t 4900 d. was employed. While the maximum absorption of light occurs between 4700 and 4800 A., as innicated in Figure 2, the 4900 A. filter gave satisfactory results. The quantity of mercury present is obtained from the curve (Figurre 3) in which the photometric densities (2 - G) are plotted on the ordinate axis and the micrograms of mercury on the
VOLUME
496
19, N O . 7
RESULTS
Table 11. Recoveries of Known Quantities of ?Zercury from 100 MI. of Urine Mercury hlercury rldded Recoyered Error Mzcrograms Mtcrograms Mtcrogram. 2.5 4.50 +0.25 5.0 +0.25 7.00 0 00 5.0 6.75 10.0 11.50 -0 25 10.0 11.75 0.00 20.0 -0.25 21.50 30.0 -0.50 31.25 40.0 42.25 +0.50 50.0 51.00 -0.75 100.0 100.00 -1.75 Reagent blank contained 1.75 micrograms of mercury.
.
Representative recoveries from tissues and urine are given in Tables I and 11. The recoveries were done on pooled urine and blood specimens which did not contain demonstrable quantities of mercury. I t will be noted that recoveries are obtainable with an error of less than 2 micrograms. The working curve is of sufficient size to permit reading 0.25 microgram and no attempt was inadr to rst,imate smaller quantities. ACKNOWLEDGMEhT
The author is indebted to Elmrr F. Kluchrsky for aiding in the ' early part of this Lyork. LITERATURE CITED
abscissa axis. The curve it: constructed on a basis of concentration of mercury per 25 ml. However, increased sensitivity can he obtained by employing a smaller volume of dithizone. The values for the construction of the curve were obtained by extracting known amounts of mercury under conditions similar to those described in the procedure. The standard mercury solution was prepared by dissolving 10 grams of metallic mercury in approximately 200 ml. of nitric acid and then diluting to 1 liter. Subsequent dilutions of this standard were made so that 1 ml. contained 5 micrograms of mercury.
(1) Cholak, J., arid Hubbard, D. M., IND.EXG.CHEM.,A N - \ L .ED.. 18, 149 (1946). ( 2 ) Gettler, A. O., and Lehmari, R. A., A m . J . Clin. Path., Tech. Suppl., 8, 161 (1938). (3) Hubbard, D. M . , IND.ENG.CHEM.,A N ~ LED., . 12, 768 (1940). (4) Laug, E. P., and Eelson, K. W.,J . Assoc. Oficiel Agr. Chem., 25, 399 (1942). ( 5 ) Winkler, W.O., Ibid., 21, 220 (1938). AIDEDby
d
grant from the Wisconsin .ilurrlni Kesenrch Foundation.
Chemical and Microbiological Differentiation of Enantiomorphs of Galactose and Xylose JOHN W. APPLING, EVELYN K. R..ITLIFF, AND LOUIS E. WISE The Institute of Paper Chemistry, Appleton, W i s .
Munson-Walker cuprous oxide reducing values of L-xylose are markedly lower than are those of D-xylose. Similar differences were noted in the case of L- and D-galactose. This indicates an asymmetric oxidation of the enantiomorphs as reported by Richtmyer and Hudson. The yeast Saccharomyces carlsbergensis, which ferments D-galactose quantitatitely, is without effect on L-galactose, thus permitting a differential fermentation of the former. The yeast Hansenula suaveoEens (N.R.R.L. 838), which quantitatively ferments n-xylose, is without action on either L-xylose or L-galactose.
I
K 1936 Richtmyer and Hudson ( 4 ) show.ed that the
1)- m t i Lforms of altrose, when oxidizrd with alkaline ferricyanide or a copper tartrate solution prepared from meso- or dl-tartaric acid, showed no difference in reducing power. Similar results were obtained with D- and L-arabinose. On the other hand, the relative reducing powers of the enantiomorphs towards optically active reagents-e.g., an alkaline copper tartrate prepared from tltartaric acid-were markedly different. This indicated that such optically active reagents caused an asymmetric oxidation of the D- and L- forms of these sugars, and the enantiomorphs could be differentiated sharply by their reducing action towards such reagents. Experiments have shown that this same type of asymmetric olidation applies, respectively, to D- and L-galactose and D- and L-xylose, and that it may be measured conveniently by means of the ordinary llunson-Walker determination. Furthermore, the enantiomorphs may be differentiated quantitatively by their behavior towards certain yeasts, which ferment the D- isomer without affecting the L- isomer. Thus, Saccharomyces carlsbergensis var. mandshuricus (N.R. R.L. S o . 379), which caused the complete fermentation of D-
galactow (b),left the L- i w n t ' r unfermented. Similarly, Hansenula suaveolens (X.K.R.L. S o . 838), which fermented n-xylose way without action on L-xylose and, inciquantitatively (67, tientally, also failed to frrment L-galactose. EXPERIMENTAL
Two samples of L-galactose were obtained. One was prepared by Ernest Anderson, who isolated the sugar from the hydrolysis products of flaxseed mucilage (I). This sample ( S o . I), after repeated recrystallization from glacial acetic acid, showed [ a]y -77.8" (H20),thus indicating that some slight impurity was still present. The other sample was obtained through the courtesy of Horace Isbell of the Kational Bureau of Standards. This sample ( S o . 11) was prepared from D-galacturonic acid by reduction to bgalactonic acid, followed by further reduction (with sodium amalgam) to the sugar. I t showed a high degree of purity([.]go -80.4"). The >xylose, prepared by the oxidation of a sorbitol derivative, after recrystal1i:ation from glafial acetic acid and ethanol, melted a t 144-1445 , [ala0 -18 . The corresponding D-galactose and D-xylose were Pfanstiehl products, whose Munson-Walker reducing values were reported pfeviously ( 7 ) , and which were used in earlier fermentation experiments (6,6). The Fehling's solution was prepared using >(dextro)-tartaric acid.
