obtained, as well as the literature values, are given in Table V. Terminal and symmetrically substituted acetylenes yield only one hydration product, the methyl or symmetrical ketone, respectively. Hydration of unsymmetrical acetylenes differs in that it results in a mixture of two ketones. The data in Table V show that the procedure gives inconclusive results with these acetylenes because of the difficulty in separating the mixture of products formed. The epoxidation-hydroxamation test was used with all the acetylenic compounds in Table I and found to give negative results which are listed in column three. These observations are not unexpected in view of the weaker nucleophilic character of the acetylene than the olefin bond and the lesser availability of the pi electrons of the triple bond is reflected in their relative reaction rates of epoxidation of the acetylenes listed in Table IV ( I S ) . Epoxidation of acetylenes to produce oxirenes initially is not only slower but was also found by Schlubach and FranZen (16) to result in cleavage of the triple bond to acids of shorter chain length. Because of this difference in reactivity, peroxyacetic acid may be employed for selective epoxidation of an olefin bond that is conjugated with a triple-bond. H H Ar-CZC-
A ‘
=C-R
+ AcOOH
-+
H H AcOH
+ Ar-C=C-(!d-R
The only terminal acetylene that did not form the expected ketone was 1octyne which was transformed by the catalyst to a ketone whose dinitrophenylhydrazone and semicarbazone melting points corresponded to those listed for 3-octanone rather than 2-octanone. This may arise from the presence of a small amount of more reactive 2-octyne as impurity coupled with the low yield of derivative which is attributed to the short reaction time in hydration. Another possible route to formation of 3octanone is oxidation of the 1-octyne to a n allylic hydroperoxide which rearranges to a mixture of 2- and 3octanone in the presence of the electrophilic catalyst. The large differences in the literature values for the melting points of dinitrophenylhydrazones and semicarbazones can be attributed to geometric isomerism and to a dynamic isomerization between different crystalline forms (4, I d ) , Lower Limits of Detection. With 1-pentyne as substrate, the sensitivity of the boron trifluoricle catalyzed hydration and formation of dinitrophenylhydrazone was 6 mg. for qualitative detection and 60 mg. for characterization of t h e acetylene b y the melting point of the dinitrophenylhydrazone after purification by recrystallization. Care should be exercised in observing the color obtained from the dinitrophenylhydrazone and 2M methanolic potassium hydroxide inasmuch as the reagent may produce a brown-red color on standing. LITERATURE CITED
\d
(1) Ansell,
M. F., Hancock, J. W.,
Hickinbottom, W. J., J . Chem. SOC. (London) 1956, 911. (2) Bader, H., et al., Zbid., 1949, 619. (3) . , Berthelot. P.. Ann. 138. 245 (1866):,, I
139, 150 (1866j.
(4) Bryant, W. M. D., J. Am. Chem. SOC. 58,2335 (1930).
(5) Cheronis, N. D., Entrikin, J. B., “Semimicro Qualitative Organic And~$,” p. 323, Interscience, New York, lYi)(.
(6) Erdmann, H., Kother, F. Z., Anorg. Chem. 18. 48- (1898). ~-~ \
( 7 ) Heilman, R., Glenet, R., Compt. rend. 234, 1557 (1952). (8) Hennion, G. F., Vogt, R. R., Neuwland, J. A., J . Org. Chem. 1, 159 (1937). (9) Johnson, J. R.. McEwen. W. L., . J. Am. Chem. soc.’48, 469 (1926). (10) Kharasch, N., Assony, S. J., Zbid., 75. 1081 ~ ~ - (195.7). - . ~_ _ , (11) Nieuwland, J. A., Vogt, R. R., Foohey W. L., Zbid., 52, 1018 (1930). (12) Rakrez, F., Kirbey, A., Zbid., 76, 1037 (1954). (13) Raphael, R. C., “Acetylenic Compounds in Organic Synthesis,” p. 33, Academic Press, N. Y., 1955. (14) Zbid., p. 73. (15) Rupe, H., Helv. Chim. Acta 9, 672 (1926). (16) Schlubach, H. H., Franzen, V., Ann. 577, 60 (1952); 578, 220 (1952). (17) Sharefkin, J. G., Shvierz, H., ANAL. CHEErI. 32, 996 (1960). (18) Sharefkin, J. G., Sulzberg, T., Zbid., 32, 993 (1960). (19) Siggia, S., Zbid., 28, 1481 (1956). (20) Shriner, R. L., Fuson, R. C., Curtin, - I
~
D. Y., “The Systematic Identification of Organic Compounds,” p. 106, Wdey, New York, 1956. (21) Wagner, C., ANAL. CHEM. 19, 103 (1947).
RECEIVEDfor review August 31, 1960. Accepted November 29, 1960. Division of Analytical Chemistry, 138th Meeting, ACS, New York, N. Y., SepJember 1960. Taken from the A I . A. thesis of Edward M. Boghosian, Brooklyn College, June 1960.
Specific Microdiffusion Method for the Determination of Fluorine Based on the Lanthanum-Alizarin Complexone Color System SIR: As far as can be ascertained, the determination of fluorine by microdiffusion never before has been accomplished. The reason for this, undoubtedly, can be attributed to the lack of fluoride-resistant Conway dishes. The problem was solved, when modified plastic Conway dishes became available in 1959 (4). I n these dishes fluorine is liberated as HF at room temperature with HzSOd, diffuses, and 644
ANALYTKAL CHEMISTRY
is trapped in the center compartment with dilute NaOH. It has been shown by Belcher, Leonard, and West (2) that the cerium (111) chelate of alizarin complexone (1,2 - dihydroxyanthraquinonyl - 3 methylamine4 :N-diacetic acid) produces a colored complex with fluoride ion. I n an acetate buffer the red color of the cerium(II1)-alizarin complexone changes to the lilac-blue of the double
complex. The test is carried out in an acetate buffer at pH 4.3. The same authors have described a quantitative procedure for the determination of fluorine in fluorinated organic compounds ( 1 ) . Chelating reactions of complexone with metal cations have been investigated by Leonard and West (3). It was found that in addition to the cerium(II1) chelate of alizarin com-
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. T h e 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 a n
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 Glucose 100 105 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 100 101 100 97 Rhamnose
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