The Microestimation of Acetyl Groups , ADALBERT ELEK AND ROBERT A. HARTE, The Rockefeller Institute for Medical Research, New York, N. Y.
T
phthalein after a short boiling to remove the carbonic acid and the sulfur dioxide formed when sulfuric acid or p-toluene sulfonic acid is used as the hydrolyzing agent. In addition to the objections raised before regarding the boiling of the distillate, this plan of titration of portions is subject to the criticism that it is a fertile source of error, the normal titrimetric errors, individually small, being multiplied by the number of titrations (at least four) to yield a not inconsiderable sum. Mention may here be made of the method of Bailey and Robinson ( I ) , who saponify by refluxing the sample for several hours in 0.04 N sodium hydroxide and titrate the excess alkali with 0.01 N hydrochloric acid. While this method is applicable only to substances which do not possess acidic functions either per se or as a result of alkaline splittinge. g., lactones, lactams, and hyperacetylated sugars (see Pirie, 8)-their finding that the error introduced by the absorption of carbon dioxide is negligible is significant. The conditions they employ are such as to make this a prime consideration, due to the great avidity of alkaline solutions for carbon dioxide. It would seem then, that,if this error is negligible under their conditions, it may certainly be dis-
H E importance of possessing a completely trustworthy method for estimation of acetyl groups is emphasized by the great value of acetylated derivatives in sugar chemistry and the signzcant role which recent biochemical investigations have ascribed to acetyl groupings in substances of biollogical interest. Especially is this so in the examination of substances of unknown constitution where the result of a determination must be used for the formulation of a reliable structure for the substance. Almost all methods, including the one here described, involve a preliminary hydrolysis of the sample followed by a determination of the acetic acid liberated, either in the reaction mixture itself (which is possible only if the solution is not so colored as to obscure the transition of an indicator or where, if that condition prevails, the titration is performed electrometrically, If), or subsequent to its removal therefrom by distillation. The methods of Freudenberg and his co-workers (9,4), where transesterification in alcoholic solution and separation of the ethyl acetate formed precede saponification, and the method of Kuhn and Roth (6), where examination of the products of chromic acid oxidation is involved, are exceptional. A comprehensive review of the literature up to 1931has been made by Meyer (8). Since the appearance of the micromethod of Pregl and Soltys (IO) who used p-toluene sulfonic acid as hydrolyzing agent, there have been two significant attempts to modify this technic in order to overcome certain inherent defects. The first of these is the method of Friedrich and Rapoport (5) who essayed a simplification of the apparatus and the manipulation. The use of a small Claissen flask as hydrolyzing vessel and the omission of the U-tube make the apparatus simpler, and the replacement of the acidimetric titration by the more elegant iodometric method is a desirable improvement. However, the regulation of the air stream through the apparatus during the distillation in vacuo to so slow a rate (only one bubble in 1 to 2 seconds) is in practice extremely difficult to maintain during the successive changes in temperature in the system and seems to be very critical. The technic recommended for the removal of the carbon dioxide from the contents of the receiving flask after the first, titration is so very sensitive t h a t the introduction of an error of considerable and indeterminable magnitude by the loss of acetic acid during the boiling out is greatly facilitated. More recently the method of Kuhn and Roth (6) has been described. These authors have devised an apparatus in which degradation of the sample may be accomplished by alkaline hydrolysis in either aqueous or alcoholic solution, by acid hydrolysis, or by chromic acid oxidation. In order to use the last-named reagent it is necessary to know the structure of the compound being analyzed to account for all the groupings which might yield volatile acids. Alkaline hydrolysis more easily ruptures certain linkages but, in the absence of definite criteria for this, the use of alternative reagents seems to complicate the problem of analysis unnecessarily. Further, the well-known solvent action of alkali on glass makes for undesirable effects on the apparatus. Alcoholic solutions of the reagents are desirable under certain conditions, to provide a more homogeneous reaction mixture. However, the technic is complicated by the necessity for removal of the alcohol prior to the distillation of the volatile acid. 1)istillation in this method is conducted a t atmospheric pressure, successive portions of distillate being titrated separately with 0.01 N sodium hydroxide against phenol-
I I I I I ) I ( / ( I
ocm.
5
10
FIGURE1. DIAGRAX or APPARATUS
267
268
INDUSTRIAL AND ENGINEERING CHEMISTRY
regarded where the solution t o be titrated possesses less chemical affinity for carbon dioxide because of its acidity. In the method described below the hydrolysis is performed with aqueous p-toluene sulfonic acid in a simplified apparatus and, following distillation in vacuo, the sulfur dioxide and acetic acid are measured by iodometric titrations. While the time required for an analysis is not reduced, fulfillment of the conditions set forth guarantees accurate results in all pases except where more than one volatile acid is produced by hydrolysis. Excessively high results arise from this last condition. Duplicate analyses show that the precision of the method is high.
Experimental The apparatus, as shown in Figure 1, is constructed wholly of Pyrex gIass, with the joints especially ground t o be vacuumtight with only water as lubricant. The connection between the side arm of the flask and the condenser is glass to glass, sheathed in steamed rubber tubing in order to provide sufficient flexibility to permit tapping the flask. It is important, therefore, t h a t these parts be made of the same diameter tubing. For the hydrolysis a 25 per cent aqueous solution of ptoluene sulfonic acid is employed. I n addition the following reagents are required: 1. An approximately 0.01 N iodine solution, prepared by dissolving 1.27 grams of analytical reagent iodine in a solution of an equal weight of analytical reagent potassium iodide in freshly boiled distilled water and diluting to 1 liter (or by dilution of an 0.1 N solution). This solution need not be standard. 2. An approximately 0.01 N sodium thiosulfate solution best prepared by dilution of an aged 0.1 N solution,.preserved by the addition of 1 per cent amyl alcohol ( 7 ) . This solution must be standardized a t least a t intervals of 2 to 3 days. Standardization may most conveniently be effected by means of potassium biiodate. 3. A 0.01 N solution of potassium biiodate prepared by weighing out on an analytical balance exactly 325 mg. of the recrystallized and well-desiccated salt and diluting to exactly 1 liter with boiled out distilled water. 4. A 1 per cent solution of soluble starch in saturated sodium chloride solution. 5. A 4 per cent solution of analytical reagent potassium iodate in freshly boiled distilled water.
The whole apparatus, with the exception of the rubber connection which is well steamed, is thoroughly cleaned with chromic acid cleaning miyture, well rinsed, and dried before using. Between successive determinations thorough rinsing with distilled water is sufficient and only the flask need be dried. A well-ground and thoroughly dried sample of from 4 to 10 mg., accurately weighed by difference on a microbalance, is placed on the bottom of the hydrolyzing flask, preferably using a long-handled charging tube. The flask is then filled about three-quarters full with short pieces (4to 5 mm. each) of 4-mm. Pyrex rod. (In preliminary trials soft glass beads were used. These quickly became porous as evidenced by their opacity and were discarded in favor of the Pyrex rod which has since been used without deterioration. This has been remarked before in connection with the original Pregl halogen method, where beads were a t first used in the combustion tube and were later replaced by a spiral.) The funnel, wet on the ground joint with distilled water, is inserted and fastened with short wire springs. Two milliliters of the sulfonic acid rea ent are then added through the funnel and the closed stopcocg is sealed with 2 to 3 drops of distilled water added through the funnel. In the analysis of halogen-containing compounds, a few milligrams of silver sulfate should be added. The side arm of the flask is connected to the condenser by means of the freshly rinsed short section of rubber tubing, a glass-to-glass joint being easily effected. The receiving flask, previously charged with 5 ml. of the 0.01 N iodine solution with 1 to 1.5 grams of potassium iodide (conveniently measured out
VOL. 8, NO. 4
as a powder by means of a small marked test tube) dissolved therein, is attached to the rubber stopper on the lower end of the condenser and adjusted so that the sintered plate is about 1 om. above the surface of the liquid. (The majority of the determinations reported here were carried out using a quartz flask as receiver. Substitution of a Pyrex receiver was accompanied by no change in the results.) The side arm of the receiver is capped with a well-washed rubber nipple. The receiver, up to the side arm, is cooled by immersion in a beaker of finely cracked ice, renewed from time to time during the hydrolysis after removal of the water formed by the melting. A stream of cold water is circulated through the jacket of the condenser. The flask is tapped sufficiently to mix the sample intimately with the acid. An asbestos board is placed as a shield between the burner and the condenser. The mixture is heated by bringing the water in the surrounding beaker to boiling. The level of water in the beaker is maintained during the period of heating by using an inverted bottle containing water, held well above the apparatus, and having a 1-cm. tube reaching - just - to the desired level in'the beaker.During the course of the heating the flask is vigorously tapped a t intervals in order to agitate its contents. Hvdrolvsis is continued for 1 hour for 0-aietyl and 2.5 to 3 hours for NIacetyl compounds. Where the nature of the linkage is unknown the longer period is, of course, to be em loyed. At the end of the hydrolysis the hot water is siphonezout of the beaker and replaced by ice-cold water. After leaving the apparatus to cool for about 5 minutes, an aspirator is connected through a manometer and stopcock to the side arm of the receiver and the system is evacuated to a pressure of 50 to 60 mm. The receiver is lifted, together with its cooling beaker, until the sintered plate is about 2 mm. from the bottom of the receiver and the water surrounding the hydrolyzing flask is heated. At first a few very small bubbles are seen to pass through the iodine solution, but the bubbling stops in a few seconds a8 the pressure is equalized throughout the system. When the fluid in the flask has completely distilled over, aided by occasional tapping, about 1.5 ml. of water are run in through the funnel without either breaking the vacuum or stopping the heating. After the contents of the flask have become dry, a second and finally a third portion of water are introduced in a similar manner. Heating is then continued until the contents of the flask are completely dry and for 5 to 10 minutes thereafter. The receiver is then lowered, using a gentle rotatory motion, until the sintered plate is 2 to 3 cm. above the fluid. The stopcock between the receiver and the gage is closed; the connection between the stopcock and the gage is broken, and then by slowly opening the stopcock the system is brought back to atmospheric pressure, and the burner is extinguished. The condenser is disconnected from the flask and washed through three times with small volumes of distilled water, flow across the sintered plate being aided by application of suction through the side arm of the receiver. The outside of the lower part of the condenser is washed into the receiver with distilled water; the total volume should be about half its capacity. The solution in the receiver, whose side arm i s capped with the rubber nipple, is titrated with the standard thiosulfate, using 2 drops of the starch indicator added toward the end of the titration. To the now colorless solution about 2 ml. of the potassium iodate solution are added, and the receiver is immediately stoppered with a soda-lime tube, placed in a beaker of water at about 35' C., and allowed to stand for 20 minutes. At the end of that time the solution is titrated with the thiosulfate solution, the end point being taken where the blue color which develops during the titration just disappears. The value from the initial titration is to be subtracted from the value obtained by titration of 5 ml. of the iodine solution in which 1 to 1.5 grams of the potassium iodide are dissolved and 2 to 3 drops of dilute acetic acid added. (It is especially important when measuring the iodine solution from a microburet to wait 1 to 2 minutes before reading the buret; the drainage error may otherwise be considerable.) This latter titration should not be made until the mixture has stood for about 5 minutes a t room temperature. The difference so calculated is a correction for the sulfur dioxide and varies from 0.00 t o 0.05 ml. Where it exceeds the upper limit the analysis should be rejected. The difference, doubled t o account for the dibasicity of the sulfuric acid formed, is deducted from the final titration as a correction for the acid introduced as sulfur dioxide.
ANALYTICAL EDITION
JULY 15, 1936
269
TABLEI. REPRESENTATIVE ANALYTICALRESULTS No.
Compound
Moles of Acetyl, Nature of Linkage
Formula
VOl. 0.01 N NszSzOa Corr. M1.
Acetyl Found
Acetyl Calcd.
%
%
9,800 8.400 6.905 11.380 7.520 8.102 8.320 9.095 8.500 8.479 5.824 7.021 4.986 7.317
8.37 7.22 5.99 9.85 4.94 5.30 5.51 6.20 4.61 1.92 7.76 5.32 6.86 9.80
36.74 36.65 37.30 37.20 28.26 28.15 28.49 28.43 23.33 9.50 55.06 32.60 59.17 57.59
7.711 5.630 6.299 6.598 5.598 5.818 5.400 5.694 6.178 6.110
4.93 6.10 5.36 5.62 4.14 6.03 4.56 4.79 8.28 6.83
27.47 45.35 36.60 36.65 31.83 44.58 36.34 36.18 57.61 48.04
Weight of Sample
MQ. 1
Tetraacetyl nitropbenol-8-galactoside Triacetyl inonoacetone glucose
CzoHzzOizN ClSHZlO9
3-0
3
Triacetyl xylosazone
CzsHzsOaNi
3-0
4
Theophylline triacetyl rhamnose
CioHzipON4
3-0
CisHzzOs CzaHz7OiN CiaHizOii CnaHsiOsN~ ClSHZ6~lZ CisHzaOinN
2-0 1-0 5-0 4-0 6-0 6-0
12 13
Rexose benzylidene diacetate glucoside Benzylidene anisaldehyde acetate aminoglucoside Galactose pentaacetate Tetraacetyl galactose phenylosazone Mannitol hexaacetate Aldehydoglucose oxime hexaacetate p-Nitrobenzyl glycoside of triacetyl galacturonic acid methyl ester Heptaacetyl methyl ester of gentiobiuronic acid Aceturio acid
CzoHzsOizN CZlH36OlO CaHiOaN
3-0 7-0 I-N
14 15 16
Acetanilide p,p’-Diacetyl phesen>cIiamine . Nitro-p,p’-diacetyl phenylenediamine
CsHoON CioHiz0,Nz CioHiiOiNs
1-N 2-N 2-N
17 18
@-Glucoseoxime hexaacetate @-Glucosesemicarbazone pentaacetate
2
5 6 7 8 9 10 11
---
. CisHzsOirN CirHzaOiiNa
The acetyl content is given by the formula
% CHsCO
= 100 X
volume of thiosulfate (corr.) X factor of solution X 0.4302 weight of sample
Representative analytical results are presented in Table
I,
Discussion The method described here is calculated to bring microacetyl determinations, which have hitherto been regarded as not entirely satisfactory for general use, into line with the more common organic microanalytical procedures as regards accuracy and precision. The distillation is improved over previous methods by omitting an air stream and establishing uniform low pressure throughout the apparatus. Introduction of a sintered glass plate a t the lower end of the condenser ensures complete absorption of the distillate in the receiving fluid, while the conditions prevailing throughout the manipulation have rendered completely negligible any error which might be due to carbon dioxide. In fact, a blank analysis made with glucose required no 0.01 N thiosulfate, showing complete absence of carbon dioxide. However, these experiments were performed in a well-ventilated microanalytical laboratory, and it is readily conceivable that other results might be obtained elsewhere. While i t may appear that, in comparison with other methods, the times recommended for the hydrolysis are unnecessarily long, the employment of these conditions ensures complete hydrolysis regardless of the nature of the linkages. Since the operation proceeds with very little attention during the hydrolysis, the increase in time is unimportant, especially when the general use of the longer period may so often save the repetition of an analysis. It is true that in some cases hydrolysis is complete in 20 to 30 minutes but there is no way in which such may be identified beforehand. In fact, the /?-glucose oxime hexaacetate (compound 17 in Table I), where one acetyl is linked to nitrogen and the other four to oxygens, gave satisfactory figures with only 30 minutes’ hydrolysis. I n several cases the 1.5 hours’ hydrolysis recommended by Friedrich (5) for N-acetyl was found to be insufficient, values in concordance with theory being obtained only after 2.5 to 3 hours. Using aceturic acid (acetylglycine, compound 13 in Table I) accurate figures were obtained in 2.5 hours, while poorer results were reported by Kuhn and Roth (6) who used alkali for the same time. Freudenberg and Soff
4-0
5-0; 1-N 4-0; I - N
36.70 37.26 28.3928.50 23.50 9.70 55.10 32.62 59.41 57.69 27.49 45.30 36.75 31.85 44.76 36.27 57.69 48.08
(3) had to hydrolyze their sample 11 to 15 times to get satisfactory results. It has always been felt that quantitative distillations at reduced pressure were not suitable for microanalytical procedures, especially where the substance sought has a fairly high vapor pressure a t ordinary temperatures. The method of Kuhn and Roth (8) was designed in part to overcome the weakness of such a procedure, However, quantitative distillation of acetic acid a t atmospheric pressure is also accompanied by some uncertainty. Absence of a difficultly regulated gas stream and improved absorption of the distillate in the receiver of the sintered plate have made quantitative recovery of the distillate a routine requiring no elaborate precautions. With the apparatus and method reported here there is no necessity for the frequent changes of temperature prescribed by Friedrich (5) and the extra distillation recommended here constitutes an additional guarantee of complete removal of the volatile acid.
Summary An apparatus is described and a procedure recommended for the quantitative determination of acetyl in 4-10 mg. of substance. A method for the quantitative distillation of volatile substances in vacuo for analytical purposes is elaborated.
Acknowledgment The authors are indebted to M. L. Wolfrom, W. F. Goebel, and R. 8. Tipson for their kindness in supplying many samples. To the last the authors are grateful for permission to publish data on an as yet undescribed preparation.
Literature Cited (1) Bailey, A. J., and Robinson, R. (2) Freudenberg, K., and Harder, (3) Freudenberg, K., and Soff, K., (4) Freudenberg, K . , and Weber, (1925).
J., Mikrochemie, 15, 233 (1934). M., Ann., 433, 230 (1923). Ibid., 494, 68 (1932). E., 2. angew. Chem., 38, 280
(5) Friedrich, A., and Rapoport, S., Biochem. Z., 251, 432 (1932). (6) Kuhn, R., and Roth, H., Ber., 66, 1274 (1933). (7) Mayr, C., and Kerschbaum, E., 2. anal. Chem., 73, 321 (1928). (8) Meyer, Hans, “Analyse und Konstitutionsermittlung organischer Verbindungen,” 5th ed., p. 339 ff., Berlin, Julius Springer, 1931. (9) Pirie, N. W., Biochem. J.,30, 369 (1936). (10) Pregl, F., and Soltys, A., Mikrochemie, 7, 1 (1929). (11) Wolfrom, M. L., Konigsberg, M., and Soltzberg, S., J. Am. Chem. Soc., 58, 490 (1936).
RECEIVED March 24. 1936.
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