General Determination of Acetyl ALAN CHANEY and M. L. WOLFROM Department o f Chemistry, The O h i o State University, Columbus 70, O h i o
which had been employed for a number of years in this laboratory
An apparatus is described by which precise values for both 0-acetyl and N-acetyl may be obtained with a minimum of operator attention. The method has been applied to a variety of carbohydrate and other materials of both monomeric and polymeric types. Wettability of the sample is required.
T
(16). PROCEDURE
H E determination of acetyl has been of importance in the carbohydrate field for more than a century. A multitude of procedures has been developed (11), many of which are applicable only under very specific conditions. I n the field of high polymers, the Eberstadt alkaline saponification method ( 4 , 8 ) , the 0 s t acid distillation (8, 12), and the alcoholic alkali saponification method (8)have been found most useful, each offering certain advantages. For simple carbohydrate derivatives the analytical procedure for 0-acetyl devised by Kuna (9) and modified by others (1, 1 4 ) has been of great value. Analyses for N-acetyl have been carried out in many ways. The original Ost procedure (12) has been employed, although it has been variously modified ( 2 , 5 , 15) to avoid the use of sulfuric acid. The use of sulfonic acids, first suggested by Sudborough and Thomas ( I S ) , was extended by Freudenberg and coworkers (6, 7 ) , who distilled ethyl acetate from an alcoholic solution of p-toluenesulfonic acid. I n spite of the great variety of methods available (11), there is none which can be applied with equal accuracy to monomeric and polymeric substances, containing either or both N - and 0-acetyl groups, that does not require an inordinate expenditure of time. The method described herein was devised to provide such a general procedure which would require a minimum of operator attention. The apparatus illustrated in Figure 1 was developed to reduce the operator time for a method based on the Ost technique ( 1 2 )
A concentrated solution of p-toluenesulfonic acid (15 grams in 25 ml. of water) was placed in flask K (Figure 1) and sufficient water was admitted through H to bring the volume of solution in K to 60 ml. The heating tape wound around K was connected to a source of 110 volts and distillation was started. The mercury level in D was adjusted with F to maintain the water level in K a t the original level during distillation. The solution level in K can be held constant (within zk1.5 mm.) with this arrangement except for an extremely slow drift (never more than 2 mm. in 24 hours) toward higher levels, which is probably caused by changes in barometric pressure. If the level should decrease too far, large errors arise due to decomposition of the p-toluenesulfonic acid ( 2 , 5 ) . The condensate from condenser Jf passed through S,0, and P, and was collected in two 2-liter Erlenmeyer flasks (vented with Ascarite protection) attached to the arms of P. Under the conditions described, a rate of distillation of about 140 ml. per hour was obtained (80 to 85% efficiency based on the heat input). The insertion of a Kjeldahl-type trap at L reduced this rate by more than 50% and prolonged the determination without increasing the accuracy. Because the p-toluenesulfonic acid employed was not especially purified, the first 500 ml. of distillate collected mas contaminated u ith acidic impurities and was discarded before any determinations were attempted. The distillate collected (20 successive l-liter portions) after the first 500 ml. exhibited a constant blank, consuming 0.59 =t0.02 ml. of 0.055 sodium hydroxide per liter when phenolphthalein indicator was used, This blank was about 40% higher than that obtained when the distilled water employed was distilled in the absence of the sulfonic acid. The procedure normally followed was to distill 500 ml. of water to remove the impurities in the sulfonic acid. Then, with distillation continuing, one of the stoppers of flask K was opened momentarily, the sample was dropped in, and the apparatus closed immediately, The sample (20 to 120 mg.) was weighed to hO.05 mg., either as a compressed pellet or in a gelatin capsule. The latter method produced an increase of about 5 % in the blank, which was corrected for by employing the blank value obtained
Figure 1. Apparatus for determination of acetyl A.
B.
Siphon connection (with air bubble trap) t o distilled water supply in 20-liter bottle (supported about 0.5 meter above) arran ed to provide constant head b y extenhon of %scarite-protected air inlet t o bottom of bottle Tveon tubine connections
G. Mercury, sufficient to balance head applied a t H.
I. J.
K.
L.
M. N. 0.
P.
A Capillary, 5 cm. long, 1-mm. i.d., to reduce diffusion from flask Solution of p-toluenesulfonic acid Glass ebulator tube Three-necked (24/40 standard taper), 200-ml. conical flask with H sealed 1.5 om. through bottom. T h e bottom 5 cm. of the flask is wrapped with 2 feet of 0.5-inch heating tape (100 watts): entire flask, including joints, is insulated with several layers of woven asbestos tape Straight tube, 8 cm. long, 20-mm. bore, insulated with woven asbestos tape’ one model was constructed with large bdre Kjeldahl vapor trap Condenser with two -4llihn-type bulbs and 12 spirals in 40-mm. 0.d. X 35-cm. jacket, arranged so t h a t all condensate is delivered to side arm Stopcock, Newman type, 10-mm. bore Condenser, 10-mm. bore, 15-om. jacket length 3-Way stopcock,, 3-mm. bore, connected to alarm clock which controls direction of flow
1614
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
Compound Eiyttiritol tetraacetate
D-llannitol hexaacetatr myo-Inosaniine hexaacetatr Tetra-0-acetylgalactaric acid
Penta-0-acetyl-aldehgda-Dgalactose aldehydrol 2-Phthalimidoethyl acetate
Sample Wt., Mg. 80.1 36.2 78.2 26.1 45.7 40.9 109.6 21.3 44.8 51.4 36.8 27.1 76.0 27.2 82.1 46.7 50.1
21.4 (6.3
Table I. CHJCO, 70 Calcd. Found 59.31 59.27 59.34 59,28 59.32 59 29 59 ~.29 59.45 59.52 59.49 59.43 59.86 59.90 59.83 59.82 45.51 45.53 45.54 52.70 52.67 52.71 18.45 18.42 18.44 37.29 37.29 ~~
1615
Results of Acetyl Analyses E ~ P.P.T. -0.7 f0.5 -0.5 +0.2 -0.3 -0 3
~
fi.1
+0.7 -0.3 +0.7 -0.5 -0.7 +0.4 f0.7
-0.6 f0.2
-1.6 -0.5
Ethyl tri-0-acetyl-nL-galactarate 0 lactone 28.42 Methyl 3,4,6-tri-O-acetyl-228 47 +1.8 87.3 [ (benzyoxy)-carbonylaniino1-2deoxy-D-glucopyranoside 1,3,4,6-Tetra-O-acetyl-Z-(p77.1 36.99 37 1 1 c3 9 methoxybenzylideneamino) -2deoxy-D-glucopyranose a Commercial samples; manufacturer’s analysis. b Calculated from sulfur analysis assuming no loss of acetyl; see Cramer and C Calculated for C ~ ~ H I ~ O I ~ S N ~ Z ( ~ H C O C H J ) .
after distillation of the acid released from the sample. Thus, distillate from the sample (about 1.5 liters) was collected in one of the flasks attached at P for 11 hours, a t which time the clock connected with string to the arm of P turned P to divert the distillate to the other flask. I n the morning, both flasks were titrated with 0 . 0 W sodium hydroxide and the titer for the sample was corrected for that of the blank to obtain the true titer for the sample. Carbon dioxide was excluded from the distillates; therefore, no precautions were necessary to avoid errors from this source ( 2 , 6). I n most cases another sample could be introduced without ceasing the distillation. I n one case, six consecutive samples were determined without interruption. Hon-ever, some materials decompose and eventually the blank values increase permanently to a point too high for toleration (more than twice the original). The results obtained for a large number and variety of samples are presented in Table I. Attempts to determine the acetyl content of tetra-0-acetyl1,B-di-O-trityl-n-mannitol were unsuccessful, with only 10% of the theoretical amount of acetic acid being obtained from the distillation of several times the normal volume of distillate. This substance was not wetted by bhe acid solution and could not be made to react. DISCUSSION
An examination of the data of Table I demonstrates that the errors in the procedure described herein are of the order of less than 1 part per 1000 in most cases. Errors of this magnitude can be ascribed to errors in the titrations and weighings, because they appear to be positive as often as negative. For erythritol tetraacetate, the compound for which the largest set of values was obtained, calculations following the techniques described by Touden ( 1 6 ) indicate that the 99% confidence limits for the average (59.30%) are 59.30 i 0.10%. Thus, in 100 determinations of the acetyl content of a given sample, 99 will be within the stated range. It should be emphasized, holyever, that failure to follow the stated procedure results in grossly inaccurate analyses. The accuracy obtained herein should be considered in the light of the authors’ experience with tetra-O-acetyl-l,B-di-0-trityl-~mannitol. This compound was not wetted by the reagent solution, and the formation of acetic acid was only 10% of the theoretical amount after distillation for several times the normal duration. This finding is in harmony with the experiences of Lemieux and Purves (IO)TTith tritylated cellulose acetate, which could not be analyzed by procedures related to the one described here. Thus, as a class, those substances which are not wetted by the boiling sulfonic acid solution will not be amenable to analysis by this technique. The size Of the shown required to Obtain the depends on its acetyl content. I n most cases, the best results
~
Sample Wt.,
~
, Compound Acetanilide
Mg.
o-Ethoxyacetanilide cis-2-Acetamidocyclohexanol 1,3,4,6-Tetra-O-acetyl-2-amino2-deoxy-D-glucopyranose hydrochloride 2-Acetamido-2-deoxy-B-Dglucopyranose Z-Amino-Z-deoxy-j3-Dglucopyranose pentaacetate 8-D-Glucopyranose pentaacetate Penta-0-acetyl-D-gluconamide keto-D-Fructose pentaacetate Sucrose octaacetate Cellulose acetate A Cellulose acetate B Tosylated cellulose acetate A Sodium chondroitin sulfate
40.5 32.3 50.7 53.6 47.2 43.4 47.6
CHzCO, % Calcd. 31.84
24.01 27.38 44.86
Found 31.82 31.83 31.85 23.99 24,02 27.36 44.82
E
~
P.P.T. -0.5 -0.3 +0.3 -0.8 +0.4 -0.7 -0.9
62.3
19.46
19.45
-0.5
49.8
55.27
55.30
$0.5
26.7 45.1 30.5 46.2 39.4 37.7 41.6 100.8 42.9 98.2 57.9 57.5 120.3
55.13
55.12 55.13 53.06 53.11 55.16 50.73 50.71 31.49 31 52 39.43 39.42 21.16 8.69
-0.2 0
53.09 55.13 50.74 31.54a 39.4“ 24 25b 8.57C
-0.6
+0.4 +0.5 -0.2 -0.6
..
..
+i4
Purl-es (S) for substantiation.
are obtained if samples no smaller than 40 to 50 mg. are employed. However, samples as small as 20 mg. may be analyzed in some cases with only slightly less accuracy. Samples smaller than 20 mg. have not been used because the difference between the blank and sample titers then becomes very small. I t is possible that further refinements would permit the utilization of smaller samples. The advantages of this method in comparison y i t h that most frequently employed for N-acetyl(6,Y) are: ( a ) A higher reaction temperature is employed, thus avoiding the low results often obtained due to incomplete cleavage of the N-acetyl linkages; ( b ) the operator time and attention required is considerably less although the total time required is greater; (c) several consecutive determinations may be carried out without cleaning or dismantling the apparatus. ACKNOWLEDGMENT
This work was supported under contract DA-33-019-ord-1466 between the Ordnance Department, United States Army (Supervising Agency, Ballistic Research Laboratories, Aberdeen Proving Ground, hld.), and The Ohio State University Research Foundation (Project 589). LITERATURE CITED
(1) (2) (3) (4) (5) (6) (7)
(8) (9) (10)
(11) (12) (13) (14) (15) (16)
dlicino, J. F., ANAL.CHEX.20, 590 (1948). Bradbury, R. B., I b i d . , 21, 1139 (1949). Cramer, F. B., Purves, C. B., 4. Am. Chem. SOC.61, 3458 (1939). Eberstadt, O., dissertation, “Uber Acetylcelluloae,” Heidelberg University, Germany, 1909. Elek, A., Harte, R. A., IXD. ENG.CHEM.,ANAL. ED. 8, 267 (1936). Freudenberg, K., Harder, hl.,Ann. 433,230 (1923). Freudenberg, K., Weber, E., 2. angew. Chem. 38, 280 (1925). Genung, L. B., Nallatt, R. C., ASAL.CHEM.13,369 (1941). Kunz, A., Hudson, C. S., J . Am. Chem. SOC.48, 1978 (1926). Lemieux, R. U., Purves, C. B., Can. J . Research 25B,485 (1947). lleyer, H., “Analyse und Konstitutionsermittlung organischer Verbindungen,” 6th ed., pp. 421-37, J. Springer, Vienna, 1938. Ost, H., 2. angew. Chem. 19, 993 (1906). Sudborough, J. J., Thomas, W., J . Chem. SOC.87, 1752 (1905). Kolfrom, 11. L., Konigsberg, hl., Soltaberg, S.,J . Am. Chem. SOC.58, 490 (1936). Wolfrom, U.L., Weisblat, D. I., Karabinos, J. V., llcSeely, W.H., McLean, J., Brown, R. L., Ibid., 65, 2077 (1943). Pouden, W. J., “Statistical Methods for Chemists,” Wiley, New York, 1951.
RECEIVEDfor review M a y 3, 1956. Accepted M a y 26, 1956. Division of Carbohydrate Chemistry, 130th meeting, ACS, Atlantic City, N. J., September 1956.
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