ANALYTICAL CHEMISTRY
590 mineral was extracted from the powdered rock samples by flotation or by treatment with hydrofluoric acid. The amount of graphite used for combustion was usually approximately 200 mg. and the recovered barium carbonate amounted to 50 to 300 mg.9 or enough for the subsequent determination of the carbon isotopes. The barium carbonate precipitates obtained were free from contamination by extraneous carbon compounds.
ACKNOWLEDGMENT
This investigation nras carried out a t the Institute of Chemistry, University of Helsinki, Finland. The kindness of Antti Auterinen in supplying expert advice on combustion methods is gratefully acknowledged, RECEIVEDJ u I r 21, 1947
Microdetermination of 0-Acetyl Groups JOSEPH F. ALICINO Squibb Znstitute f o r Medical Research, New Brunswick, N . J .
T OFTEI; becomes necessary in structural investigations to Idistinguished determine accurately the number of 0-acetyl groups, as from N-acetyl groups, in a compound containing
(r),
both. The excellent method of Kunz and Hudson as modified by Wolfrom, Konigsberg, and Stoltaberg ( I O ) , accomplished this objective and is now widely used for this purpose in sugar chemistry. It is based on the fact that 0-acetyl groups are quantitatively liberated by 0.1 N sodium hydroxide in acetone solution, within a few hours a t 0' C., while N-acetyl groups are not attacked under these conditions. However, the method in its present form requires about 100 mg. of substance, an amount which cannot always be spared in investigations on rare sugars. In connection with studies conducted in this laboratory on degradation products of streptomycin, it became desirable to adapt the procedure of Kunz and Hudson to a micro scale. Because the use of more dilute alkali was imperative in such a micromodification, the completeness of saponification of mannitol hexaacetate by 0.01 N alkali a t 0 " and a t room temperature (24 ' to 27 C.) was first investigated. The results summarized in Table I indicate that satisfactory values could be obtained after 2 hours a t room temperature and that no untoward results are produced by increasing the reaction time.
Table I. Determination of @Acetyl on AIannitol Hexaacetate Temperature, 'C. 0
Room temp. (24-27')
Time, Hours 2 1
2
5
% Theoretical 60 6..7
80 99-100 99-100
PROCEDURE
The sample (4 to 10 nip.) is weighed into a 50-nil. glassstoppered flask and 5 nil. of C.P. acetone and 10 ml. of aqueous 0.01 ,I' sodium hydroxide are added. The flask is iminediately stoppered and set aside for 2 hours. At the end of this tiiiie 1 to 2 drops of phenol red are added and the solution is back-titrated with 0.01 S acid to a distinct yelloir end point which will persist for a t least 2 minutes. A blank determination on the acetone from time to time should not exceed C.2 to 0.3 ml. of (2.01 S sodium hydroxide. If the substance is precipitated by the standard solution, as happens occasionally, it is brought into solution again by the addition of another 5 ml. of acetone. When 10 ml. of acetone must be used, the blank correction is only slightly higher. Certain water-insoluble compounds, such as sterol acetates, as Fell as deeply colored subst,ances, cannot be conveniently analyzed by this method. Care must also be exercised in the interpretation of results when the substance being analyzed contains titratable basic or acidic groups, either in the free state or in a linkage from which they might be liberated by alkali under the above conditions. The results in Table 11 show that the presence of L\7-acetyl
groups, in accordance with the findings of Wolfrom et nl. ( I O ) , does not interfere. ACKNOWLEDGMENT
The author is indebted to 0. Wintersteiner for his advice and suggestions, as well as to J. Fried and Homer E. Stavely for their kindness in supplying samples for this work, and to Anne C. Crickenberger for carrying out many of the analyses.
Table 11. Typical Analyses 0-Acetyl
Compound Acetanilide Mannitol hexaacetate
@-Fructosepentaacetate @-Glucosepentaacetate Streptamine hexaacetate (8)
a-Hexaacetyldihydrostreptobiosamine (9) 8-Hexaacetyldihydrostreptobiosamine (9) a-Pentaacetyldihydrostreptobiosamine(9) a-Ethyl tetraacetylthiostreptobiosaminide diethyl mercaptal (5) @-Ethyltetraacetylthiostreptobiosaminide diethyl mercaptal (4) a-Methyl pentaacetyldihydrostreptobiosaminide ( 5 ) a-Methyl tetraacetylstreptobiosaminide dimethyl acetal ( I ) a-D-Glucosamine pentaacetate p-D-Glucosamine pentaacetate n-,~-methyl-L-gIucosamine pentaacetate (6)
Found 0.0 59.2 59.5 59.3 59.2 55,5 55.3 54.8 55.0 39.2 39.0 36.5 35.9 31.2 31.8 20.3 20.4 20.4
Calcd. 0.0 59.5
30.4
30.6
55.1 55.1 40.0 36.4 36.4 31.3 19.7 19.7
21.6 22.8 21.9 46.2 45.9 46.0 45.9 46.9" 42.7 45.9 @-31ethyltetraacetyi-D-mannopyranoside ( 2 ) 48.3 47.5 a-Methyl tetraacetyl-D-mannopyranoside (2) 4 7 . 7 47.5 a-Thioethyl tetraacetyl-D-mannoside ( 2 ) 44 2 43.8 p-Thioethyl tetraacetyl-D-mannoside (a) 43.4 43. 8 a Results on this comoound as well as other 0- and N-acetvlated derivatives of S-methylgluco~amineinvestigated after this paper had been submitted were consistently high. T h e cause of the apparent instability of t h e S-ace&l group under these conditions is being investigated.
LITERATURE CITED
(1) Brink, N. G., Kuehl, F. d.,Jr , Flynn, E. H., and Folkers, K., J . Am. Chem. SOC.,68, 2557 (1946). ( 2 ) Fried, J., and Stavely, H. E., I b i d . , 69, 1549 (1947). (3) Fried, J., and Wintersteiner, O., Ibid., 69, 79 (1947).
(4) Hooper, I. R., Klemm, L. H., Polglase, W. J., and Wolfrom, M. L., Ibid., 68, 2120 (1946). (5) Kuehl, F. il., Jr., Flynn, E. H., Brink, N. G., and Folkers, K., I b i d . , 68, 2096 (1946). (6) Kuehl, F. A., Flynn, E. H., Holly, F. W., Mosingo, R., and Folkers, K., Ibid., 68, 536 (1946). (7) Kuns, A., and Hudson, C. S., Ibid., 48, 1982 (1926). (8) Peck, R. L., Hoffhine, C. E., Jr., Peel, E. W., Graber, R. P., Holly, F. W., Mosingo, R., and Folkers, K., I b i d . , 68, 776 (1946). (9) Stavely, H. E., Wintersteiner, O., Fried, J., White, H., and Moore, M., Ibid., 69, 2742 (1947). (10) Wolfrom, M. L., Konigsberg, M., and Soltsberg, S., Ibid.. 58, 490 (1936). RacgIyEn September 23, 1947.
