plexone, only lanthanum and praseodymium chelates give a specific color reaction with fluorine ion. KO further tests were made to determine the suitability of either for the determination of fluorine. Mechanism of the reaction for the formation of the fluorine complex was proposed. h preliminary study of the lanthanum-alizarin complexone chelate for the determination of fluorine was started by the writer several months prior to the publication by Leonard and West ( 3 ) . Incomplete evidence indicates that the lanthanum chelate possesses some advantages over the cerium(II1) chelate for the determination of fluorine. The fluoride test is carried out in an acetate buffer a t pH 5.4. It is extremely sensitive and as little as 0.25 pg. can be detected. Relatively high
concentrations of the common anions do not interfere, and none of these anions produce false colors. The color change is from wine-red to lilac-blue. The solution from the center compartment is removed, the p H is adjusted to 5.4, and equal volumes of 0.001N alizarin complexone and 0.001M La(NO& are added. The solution is diluted to a suitable volume, mixed, allowed to stand for a t least 1 hour, and the absorbance is measured against a blank containing only reagents on the spectrophotometer a t 615 mp. Preliminary results indicate satisfactory precision and recoveries. This investigation is still in progress, and a comprehensive report covering every detail of the method will be published later. The cerium(II1) chelate of alizarin complexone has been used in this
laboratory for the past 6 months as a routine screening test for the detection of toxicologically significant fluoride concentrations in tissue. The recovery of fluorine from various tissues is now being investigated, and the results will be published later. LITERATURE CITED
(1) Belcher, R., Leonard, M. A., West, T. S., J . Chem. SOC.1959, 3577. (2) . . Belcher. R.. Leonard. M. A..' West.
T. S.,Tulantu 2, 92 (1959). (3) Leonard, M. A., West, T. S., J . Chem. SOC.1960, 4477. (4) Obrink, K. J., J. Biochem. 59, 134 (1955). FRANCIS J. FRERE Toxicology Section Office of Medical Examiner Philadelphia, Pa.
RECEIVED for review November 21, 1960. Accepted February 24, 1961.
Estimation of Sugars in Paper Chromatograms and Paper Electrochromatograms Sprayed with Ammonium Molybdate SIR: A previous communication (4) suggested a reagent composed of 10% ammonium molybdate for spraying paper chromatograms or paper electrochromatograms of reducing sugars. In both cases the reducing sugars are revealed as yellow spots which turn blue with time. A procedure for estimating colorimetrically the sugars revealed on chromatograms or electrochromatograms sprayed with the above reagent consists of measuring the blue coloration formed when the filter paper circles containing the sugar spots are heated with ammonium molybdate in dilute sulfuric acid. The reduction of ammonium molybdate to molybdenum blue has long been used for the detection and estimation of sugars. Thus, ammonium molybdate and hydrochloric acid were used by Matthews (6) for estimating sucrose. This reagent, however, produced rather turbid solutions, presumably due to the precipitation of molybdic acid. The addition of ammonium salts -for example, ammonium chloride, as in Aronoff and Vernon spraying reagent (I)-produces much clearer solutions, although of weaker intensity. On the other hand, replacing hydrochloric acid by sulfuric acid as in the present method leads to clear solutions even in the absence of ammonium salts. In this case, a small crystalline deposit is formed, which adheres strongly on the walls of the tube and thus enables transfer of the clear solution into the cell.
PROCEDURE
REAGENTA. Aqueous ammonium molybdate (10%) used as spraying reagent to reveal the sugar spots on paper chromatograms and paper electrochromatograms. REAQENT B. Ammonium molybdate in dilute sulfuric acid used for colorimetric estimation of the sugars in the revealed spots. Add 17 grams of ammonium molybdate to 700 ml. of water, then add 17 ml. of concentrated sulfuric acid, allow to cool, and dilute to 1 liter, Standard Solutions. For each estimation two standard artificial sugar mixtures were used, having the same components as the mixture to be separated and estimated. The concentrations of the sugar components in the first were 2% for each sugar and in the second 6% for each sugar. Any mixture of reducing sugar which can be satisfactorily separated on a paper chromatogram or paper electrochromatogram can in principle be estimated, if the concentration of each component lies between 1 and 10%. Examples of such separation and estimation are given in Table I. APPLICATION OF SPOTS. The spots of the two standard mixtures and of the mixture t o be separated and estimated were applied by means of a Kirk transfer-t.ype micropipet (2). Drops of 2.5 pl. of the standard solution (containing 50 and 150 pg. of each sugar in the first and second standard, respectively) and
5 pl. of the mixture to be analyzed were spaced regularly on the filter paper. PAPERCHROMATOGRAMS. Descending paper chromatograms were run on Whatman No. 1 filter paper (33 cm. long and 22 cm. wide) and developed with the upper layer of a mixture of n-butyl alcohol (40 m1.)-water (50 m1.)-ethyl alcohol (10 ml.) for 48 hours. PAPER ELECTROCHROMATOGRAMS. For paper electrochromatograms an
Table 1. Estimation of Sugar Components of Some Binary Mixtures
[Ethyl alcohol (10)-butyl alrohol (40)water (50)] Sugars in Taken, Found, Mixture Pg. rg. Mixtures Separated by Paper Chromatography Glucose 100 105 Arabinose 100 105 100 105 Glucose Xylose 100 95 Galactose 100 100 Fucose 100 100 Arabinose 100 93 Xylose 100 98 Mixtures' Separated by Electrophoresis Glucose 100 100 Rhamnose 100 100 Xylose 100 101 Rhamnose 100 97
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apparatus similar to that of Durrum (S), with Whatman No. 1 filter paper (30 cm. long and 15 cm. wide) was used, The electrochromatograms were run for 2 hours with 400 volts; either 1% borax solution or p H 8.6 borax-boric acid-NaC1 was used as buffer. REVEALING SUGARSPOTS. The paper chromatograms or paper electrochromatograms were dried in an oven, sprayed with reagent A and then heated for 20 minutes at loo", when the sugar components were revealed as yellow spots on a colorless background. Care was taken not to leave the chromatograms or paper electrochromatograms exposed t o strong light, since they tended to acquire a blue coloration. DEVELOPINGCOLOR FOR ESTIMATION. Circles 2 cm. in diameter were cut around each revealed spot and introduced in a test tube (25-mI. capacity), 6 ml. of reagent B were added, and the tubes were heated in a briskly boiling water bath for 15 minutes with occasional shaking, then kept a t room temperature for 5 minutes. A colorless blank was simultaneously prepared by
cutting a circle of the same size and subjecting it to the same treatment. MEASUREMENT OF COLOR. The color was measured on either a Unicam SP 500 spectrophotometer (6) at wave length 700 mp or with a Hilger Spekker absorptiometer (6) using Ilford spectrum red filter No. 608 (approximate peak of transmittance a t wave length 680 mp). In both cases, 1-cm. cells were used. DETERMINATION O F CONCENTRATION. Curves of the absorbance were plotted against weight of sugar for the two standard solutions and from the absorbance of the sugar components in the analyzed mixture, their weights were determined. DISCUSSION
Apart from its simplicity, the suggested procedure gives reasonably reproducible results. Thus, the usual error involves differences of about 5 pg. in 100-pg. samples of sugar (Table I). The number of components to be esti-
mated and their nature are limited only by whether they can be satisfactorily separated by paper chromatography or paper electrophoresis. The solvent used for the former does not seem to have any great influence, since it is evaporated. LITERATURE CITED
(1) Aronoff, S., Vernon, L. P., Arch. Biochem. 28,424 (1950). (2) Bl;ck, R. T., Durrum, E. L., Zweig, G., Manual of Paper Chromatography and Paper Electrophoresis," p. 57, Academic Press, New York, 1958. (3) Durrum, E. L., J. -4m.Chem. Soc. 72,2943 (1950). (4) El Khadem, H., Hanessian, S., ANAL. CHEM.30, 1965 (1958). (5) Rlatthews, Maryland Acad. Sci. Bull. 7, 35 (1928). (6) Vogel, A. I., "Quantitative Inorganic Analysis," pp. 621, 629, Longmans, Green, London, 1953. HASSAN EL KEADEM Chemistry Department WISSAMGIRGIS Faculty of Science
Alexandria University Alexandria, Egypt, U.-4.R.
Determination of Ruthenium Tetroxide SIR: -4study of the kinetics of the decomposition of ruthenium tetroxide necessitated determination of the quantity of RuO4 present in sealed Carius tubes containing, in addition to RUO4, other materials such as ruthenium metal, ruthenium dioxide, silica gel, and Anhydrone. Available methods measured the total ruthenium content and were not applicable to this type of sample, since ruthenium dioxide was one of the products of decomposition. Accordingly, a procedure was developed to measure RuO4 content selectively. Sandell reported (6)that ruthenium tetroxide may be extracted with carbon tetrachloride, but the stripping of the RUOa from the solvent is a slow procedure, requiring as much as 2 hours' shaking with sulfurous acid for a quantitative transfer. Marshall and Rickard (1) published a method for determining ruthenium, utilizing the absorbance of the orange-red color of the ruthenate solution. The combination of carbon tetrachloride extraction and colorimetric procedures provided the basis for the present method. The main innovation is the stripping of the tetroxide from the carbon tetrachloride using a solution of potassium hydroxide. Reagents. Ruthenium metal and ruthenium dioxide were obtained from the A. D. Mackay Co. The oxide was ignited at 950' C. to remove volatile impurities. Spectrographic analysis of the ignited sample indicated only trace impurities. The 646
ANALYTICAL CHEMISTRY
RUO4 was synthesized from RuOl via a persulfate oxidation, followed by drying with Anhydrone. Experimental. Capillary micropipets were used to transfer the liquid RuOd to the Carius tube. RuO4 was melted and an appropriate volume was drawn into the pipet with a syringe. The filled pipet was then weighed, and the RuOl was quickly transferred into a Carius tube which was chilled a t the bottom. Any R u 0 4 that remained in the capillary was frozen to reduce volatilization losses by touching the chilled wall of the Carius tube. The pipet was removed and reweighed, and the weight of Ru04 transferred into the Carius tube was obtained by difference. These tubes were sealed and used in a study of the decomposition of To determine the amount of RUO4 not decomposed, one end of the sealed Carius tube was placed in a dry iceacetone bath and the other was warmed with a hair dryer, so that the Ru04 condensed in the cold end of the tube. The top of the tube was cracked, using a hot wire, and opened. Fifteen milliliters of Cc14 was added to the tube, which was then allowed to come to room temperature. The cc14 and the tube contents were transferred with CCl4 washing to a centrifuge tube and centrifuged. The centrifugate was added to a beaker containing 50 ml. of 2N KOH. The residue (RuOz and Anhydrone) in the tube was crushed with a glass rod and re-extracted with CC14. The CCl, extract was added to the original centrifugate. The mixture of CCl4 and alkali was warmed a t about 70" to 75" C. until the cc14 layer was clear
(about hour). The mixture was transferred to a separatory funnel, the two layers were separated, and the CC1, was discarded. The aqueous layer was transferred to the original beaker and reheated to 80' to 90' C. for about 15 minutes. The KOH solution was transferred to a 250-ml. volumetric flask, cooled, and made up to volume with 2N KOH. The absorbance was then measured a t 465 mp with the Beckman DU spectrophotometer. RESULTS AND CONCLUSIONS
The combination of carbon tetrachloride extraction and KOH stripping gave the advantages of high selectivity of the solvent and development of the ruthenate color in a short period of time. The RUOc n-as rapidly extracted by the carbon tetrachloride and presented no problems. A number of tests were conducted to determine optimum conditions for stripping the RuOc from the CC14; 2N KOH a t about 70" C. gave the best results. The relatively rapid stripping with KOH can probably be traced to a combination of factors. In the procedure described, the CCl4 extract is added to a beaker containing warm KOH and is heated. This increases the hydrolysis rate of the RuOc in the aqueous phase, thereby favoring the stripping. The RuOl is also more volatile a t the higher temperature. As it volatilizes, it is absorbed in the layer of KOH and hydrolyzes. There is no appreciable loss during this step. The accuracy of the method was
