282
M.L. WOLFROM AND LOUISW. GEORGES [CONTRIBUTION FROM THE CHEMICAL
LABORATORY OF THEOHIO
VOl. 59
STATE UNIVERSITY]
A Study of Cellulose Hydrolysis by Means of Ethyl Mercaptan' BY M. L. WOLFROM AND LOUISW. GEORGES~ I t is difficult to obtain exact information concerning the structure of the cellulose molecule. The classical methods of organic structural determination involve the study of the breakdown of the organic molecule by degradative methods. The cellulose molecule may be degraded by acid hydrolysis and any new information obtained by a study of this hydrolytic process should be of service eventually in interpreting the true structure of the cellulose molecule. The recent studies of Freudenberg and of Freudenberg and Kuhn3 on the hydrolytic breakdown of cellulose by sulfuric acid and by acetolysis constitute some of the more important recent contributions to the problem of the constitution of cellulose.
first break in the curve of all celluloses examined a t a specific rotation of 4-43' instead of 14' and found in addition a second break at a specific rotation of +73O. Their temperature of hydrolysis was 20'. It is probable that variations in the type of this polarimetric curve can be ascribed to different methods of cellulose purification. We have followed polarimetrically the hydrolysis curve a t 23' of a sample of chemically purified, high viscosity cotton linters, employing solution concentrations and tube lengths to ensure adequate observed rotations. In agreement with previous work, we find that the initial rotation is zero. With the @-glucosechain conception of the cellulose structure, it is rather difficult to understand why this initial rotation should be zero rather than levo. We did not find any initial induction period, nor did we find any breaks in the curve (Fig. 1). Many intermediate readings other than those shown were obtained. Data were obtained for two concentrations of cellulose and are tabulated in Table I. An analysis of the data (Table I) shows that the calculated mono0 200 400 600 800 molecular velocity constants show a slight but Time, min. definite upward drift. The average value of the Fig. l.--Specific rotation and viscosity of 3% soluvelocity constant for the greater part of the retion of cellulose in fuming HCl 1.203) at 23'. action is practically independent of the initial In 1913, Willstatter and Zechmeister4 prepared cellulose concentration. The polarimetric data solutions of cellulose in fuming hydrochloric acid so obtained verify the results of Hibbert and and followed the hydrolysis a t 16' by polarimetric Percival,6 who followed polarimetrically the and copper reduction methods. With a cellulose hydrolysis of cellulose (alkali-purified) in a hydropreparation that was apparently only solvent- chloric acid-zinc chloride medium. These workextracted, they obtained an initial induction ers likewise obtained no breaks in the polariperiod and an inflection point a t a specific rota- metric curve and found that the calculated monotion of + 1 4 O . In 1923, Sherrard and Froehlke6 molecular velocity constant exhibited a definite in identifying some purified wood celluloses, upward drift. Their average velocity constant demonstrated their similarity to cotton cellulose value is in excellent agreement with the one by applying the polarimetric method of Will- determined in this Laboratory, employing a fumstatter and Zechmeister. They also made a run ing hydrochloric acid medium at the same temon cotton cellulose as a control. They found the perature. (1) Presented before the Division of Cellulose Chemistry at t h e In agreement with previous workers, we find 92d Meeting of The American Chemical Society, Pittsburgh, Peonthat the final rotation DI.[( 100') is approxisylvania, September 7, 1936. (2) Du Pout Cellulose Research Fellow. mately that of an equivalent amount of d-glucose. (3) K. Freudenberg, "Tannin-Cellulose-Lignin," Verlag J. The equilibrium rotation of d-glucose ia proSpringer, Berlin, 1933. (4) R. Willststter and L.Zechmeister, Bcr., 46, 2401 (1913). foundly affected by the hydrogen chloride concen( 5 ) E C Sherrard and A. W. Froehlke, THISJOURNAL, 45, 1729
+
+
1923)
(6)
H. Hibbcrt and E. G . V. Percival, ihid., 69, 3996 (1930).
Feb., 1937
A STUDY OF CELLULOSE HYDROLYSIS BY MEANS OF ETHYL MERCAPTAN
283
The main object of our investigation was to insert a carbonyl reagent into the hydrolytic mixture in order to follow the breakdown of the cellulose molecule by means of the intermediate reaction products. Ethyl mercaptan is a suitable 100 reagent for this purpose. This substance will react with the free reducing groups of sugars in concentrated hydrochloric acid to form the sugar mercaptals or thioacetals.' At the present stage of our investigations, the mercaptalated cellulose hydrolytic products, other than glucose ethyl mercaptal, were isolated as amorphous mixtures and no attempt was made to separate them into their pure components. They were characterized 0 10 20 30 40 by means of their sulfur content. Concentration of HC1, %. The reaction between a reducing sugar and Fig. 2.-Optical rotation of glucose in HC1 a t 25". ethyl mercaptan takes place with extreme rathis rotation change is reversible under the condi- pidity and at low temperatures. Under the tions of time and temperature employed and Willstatter-Zechmeister hydrolytic conditions, accordingly it must represent a true tautomeric the effective sugar concentration is relatively low and we found it necessary to study the mercapshift. talation of d-glucose under such conditions. A TABLE I MUTAROTATION OF CELLULOSE IN FUMING HCL; VELOCITY 5% solution of d-glucose in fuming hydrochloric acid can be mercaptalated by vigorous mechanical CONSTANT, TEMPERATURE 23 " shaking with an excess of ethyl mercaptan for c, 1.004; I, 4-dm. c, 2.857; I , 4-dm. 1" UD [U]D 'a OLD Ia1D fifteen minutes at 0'. At the end of this period (min.) deg. deg. kb (rnin.) deg. deg. kb the solution has lost its Fehling reduction ca19 +0.16 +4.0 0.0021 26 +0.65 f 5 . 7 0.0022 36 0.98 8 . 6 .0024 pacity and the glucose diethyl mercaptal may be .19 4.7 .0020 24 51 1.36 11.9 .0024 isolated in approximately eighty per cent yield. 44 .41 10.2 .00!24
tration. This variation is plotted in Fig. 2 and is in fair agreement with previous data. In agreement with Willstatter and Ze~hmeister,~ we find that
c
81 2.22 19.4 ,0025 18.9 .0026 29.9 .0030 101 2.80 24.5 ,0026 37.1 .0031 126 3.48 30.4 .0027 156 4.26 37.3 ,0028 44.8 .0033 49.1 .0034 186 5.02 43.9 .0029 51.8 .0033 218 5.6n 49.0 ,0029 247 6.40 56.0 ,0031 60.3 .0036 297 7.38 64.6 ,0032 64.7 ,0036 71.7 ,0038 339 8.15 71.3 ,0034 78.9 .0040 454 9.77 85.5 .0037 544 10.66 93.3 .0041 86.9 .0043 95.9 .0051 694 11.54 100.9 .0048 a 100.1 co 104.6 a The initial time was determined here by the backward extrapolation of the Curve to zero rotation. k = 1 a0 am - In, (tinminutes; 00 = 0). 79 119 149 179 199 220 259 286 334 391 478 626
t
.56 1.20 1.49 1.80 1.97 2.08 2.42 2.60 2.88 3.17 3.49 3.85
at
a,
We were also interested in following the viscosity changes in the cellulose solutions. We found that a t 25' the first measurable viscosity value was quite low and that the solutions attained the viscosity of an apparent equivalent amount of dglucose when the specific rotation was approximately + 2 5 O . These data are shown in Figs. 1 and 3.
so
120 160 200 Time, min Fig. 3.-Cmnparison of vimsity at 26": 0, 3% solution of cellulose in fuming HC1; A, 3% solution of glucose in fuming HCI; 0 , fuming HCl. 40
The glucose mercaptal that separates, after sodium bicarbonate neutralization, is contaminate d with appreciable amounts of sodium chloride. For quantitative purposes, it was found desirable to leave this salt in the product and correct for its presence by an ash determination. We are probably dealing here with an addition compound (7) E. Fischer, B o . . 97, 673 (1W4).
&I. L.
284
WOLFROM AND
similar in nature to the well-known d-glucosesodium chloride compound. The mother liquor could then be investigated by concentration to dryness and acetylation, the acetylated product being purified by pouring into a large volume of water. Difficulties were here encountered in acetylating quantitatively, a small amount of material adhering to a relatively large amount of crystalline salt that was insoluble in the acetylating mixture. The optimum mercaptalation procedure thus established for d-glucose was then applied to the cellulose hydrolytic solutions and the course of hydrolysis of cellulose (5%) in fuming hydrochloric acid solutions a t 16' was studied by means MERCAPTALATED PRODUCTS
Bxpt.
a b C
d e
f E h
i j k 1 m n
Time of hydrolysis
17 min. 3 hrs. 6 9 12 15 18 21 24 27.5 34.5 38.7 51.7 55.0
ISOLATED PROM
[u]I'D
.. +17.5" 36.7 46.6 61 .O 70.1 79.8
.. 91.5 99.8 103.0 104.9
..
w.GEOKGES
LOUIS
of the mercaptalated products obtained from the reaction of ethyl mercaptan with the cellulose solutions at various stages of hydrolysis. Simultaneous polarimetric data were obtained. Sulfur analyses served to determine the average molecular size of the mixtures of mercaptalated products isolated from the cellulose hydrolysis. The pertinent data are tabulated in Table 11. An analysis of these data is given in Table I11 and shows that the cellulose molecule fragments with extreme rapidity under these conditions. Glucose ethyl mercaptal first appears when the hydrolysis is approximately two-thirds completed. Its amount gradually increases. When the hydrolysis was approximately at the half-period, the
TABLE I1 CELLULOSE SOLUTIONS 1.203) AT 16 a
(6,
4.669)I N FUMING HYDROCHLORIC ACID (d",
Water-insoluble mercaptalated products (from 2.80 g. cellulose) Wt. 7s (NaCI-free (Ne&-free % NaCl basis), g. basis)
19.4 0.5 0.6
VOl. 59
2.7 1.5 0.7
0.33 2.03 2.39
Acetylated water-soluble products Wt. s,
Expt.
a' b'
Trace 1.1
c'
1.8 3.5 1.6 2.9 2.2 1.4 2.7 2.3 1.2
d' e'
0 5.8 6.4 9.5 17.9 18.8 11.4 20.9
0.4 1.1 1.7 1.8 2.1 2.5 2.5 2.4
f' 21.3 21.1 21.9 21.6 21.1 21.4 21.7
E' h'
if j'
k' 1' m'
%
4.6 6.7 7.4 6.0 6.2 6.0
5.7 7.1 6.8 7.7
1.3 6.2 n' 1.3 6.7 The addition of 5 cc. o f water preceding the mercaptan addition was omitted. (a) (d to n) The acid solution was poured into a suspension of 68 g. of sodium bicarbonate in 100 cc. of water. (d, e) No product precipitated. fJ) After standing for one day, 0.4 g. of a crystalline product separated. This material was characterized as dglucose diethyl mercaptal. On recrystallization the substance melted at 127"(unchanged on admixture with an authentic specimen of d-glucose diethyl mercaptal). (g to n) Crystalline products were obtained which were mixtures of glucose diethyl mercaptal and sodium chloride. They were removed by Gltration and were not washed. The sodium chloride content was determined by conversion to sodium sulfate in a platinum crucible with concentrated sulfuric acid. Calcd. for d-glucose ethyl mercaptal: S, 22.4. The glucose ethyl mercaptal was isolated and identified as its pentaacetate for one typical run. A portion (1.5 9 . ) of the crude water-insoluble product from (k) was acetylated overnight with acetic anhydride (10 cc.) and pyridine (5 cc.) and poured into ice and water (400 cc.); yield, 1.6 g. On recrystallization from methanol-water the substance was identified as d-glucose diethyl mercaptal pentaacetate; m. P. 45-47' (mixed m. p. unchanged); [ C X ] ~ * +13.3" D (c, 3.6; CHCb). (jto n) A second solution of cellulose (30 9.) was prepared as previously described and used after it had been allowed to hydrolyze a t 16' for twenty-seven and one-half hours. (a') A trace of amorphous material was obtained. (b') A solid, amorphous product was obtained. (c', d ' ) These products separated as gummy masses contaminated with salt. They were taken up in chloroform and the extract washed with sulfuric acid (1 molar), aqueous sodium bicarbonate and water. On solvent removal and trituration with ether the products were obtained as granular solids. (e' to h') The products separated as, viscous sirups which hardened to a solid mass on standing. (i' t o 9 1 ' ) These sirupy products were treated as descri1)rtl for ( c ' ) and ( d ' ) and were olltained as glassy solids aftrr i l r y i n g in a v a c u i m desiccator.
..
A STUDYOF CELLULOSE HYDROLYSIS BY MEANSOF ETHYL MEKCAPTAX
Feb., 1937
isolated mixtures of products corresponded to the of this typeof trisaccharide stage. The amount materia' gradually decreased but quantities were present at the last point determined.
285
lution after three hours of standing was found to be 1.2003; 25"/4". This made a 1% solution of cellulose (moisturefree basis) in fuming hydrochloric acid. A portion of the solution was used for the polarimetric observations and another portion was used for the direct determination of viscosity in an Ostwald type viscometer. A similar procedure was used in obtaining data for a 3% cellulose conTABLE 111 centration. The results obtained for one typical cellulose MOLECULAR SIZEOF ISOLATED MERCAPTAL PRODUCTS concentration are shown in Fig. 1. The data shown in 5% Cellulose in fuming hydrochloric acid; temp., 16'. Fig. 3 were obtained for comparative purposes. The calEach experiment equivalent t o 2.80 g. cellulose. culated velocity constant data are tabulated in Table I . Water-insoluble Water-soluble Time of product product as acetate The optical rotations of d-glucose in various concentrations hydrolysis, Amount, Amount, of hydrochloric acid are shown in Fig. 2. Hxpt. hours [alien g. D.p.0 g. D.p . a Reversibility of the Optical Rotation of &Glucose in '/4 .. 2 . 7 121 ... ... Concentrated Hydrochloric Acid.-The experiment to be b 3 $17.5" 1 . 5 20 1.1 4 described was performed to determine whether the rotac 6 36.7 0 . 7 17 1.8 2.5 tion of d-glucose in fuming hydrochloric acid was due to a a 9 46.6 0 . 3.6 2.5 permanent change in structure or to a reversible tautomeric e 12 61.0 0 . 1.6 3 equilibrium. d-Glucose (1.06% 9.) was dissolved in f 15 70.1 0.4 1 2.9 3 fuming hydrochloric acid (d16r 1.202) to make 50.00 cc. of g 18 79.8 1.1 1 2.2 3 solution; [ Q ] D +100.3". This solution was then diluted h 21 .. 1.7 1 1.4 3 to 99.90 cc. with water. This lowered the hydrogen chloi 2 4 91.5 1.8 1 2.7 2.5 ride percentage to approximately 20.1% and the concenj 27.5 99.8 2.1 1 2.3 2.5 tration of d-glucose from c, 2.125 to c, 1.063; [ a ] D +57". k 34.5 103.0 2.5 1 1.2 2 The specific rotation for d-glucose in acid of the same conm 51.7 .. 2.5 1 1.3 3 centration was found to be +56'. This indicates that the n 55 ,. 2.4 1 1.3 2.5 equilibrium is essentially reversible and that the rotation ' D. p. = degree of polymerization or number of glucose changes are measures of true tautomeric shifts. iinits in the chain. For (a), (b) and (c), this was calcuMercaptalation of a 5% d-Glucose Solution.-&Glucose lated by the following close approximation: d. p. = (3.3 8.) was dissolved in GO cc. of hydrochloric acid (@a4 100 x 2 s 40 . ~____ 1.184) and the solution cooled to 0". Purified ethyl mer%S X CsHieOs %S captan (8 cc.: cooled to 0 " ) was added and the mixture vigorously shaken by mechanical means for fifteen niinutes Further work is in progress in this Laboratory. a t 0". The faint yellow solution was poured with stirring into a suspension of 60 g. of sodium bicarbonate in 100 cc. Experimental of water. The heat change was negative and the neutraliCellulose Characterization.-The cellulose used through- zation could thus be effected a t low temperatures. The out this research was a sample of purified cotton linters8 of mixture was cooled in an ice-snlt bath and the crystalline high viscosity which had been subjected to the customary product removed by filtration, washing being omitted; alkali kier and mild bleaching process. The material yield, 4.9 g. or 4.0 g. on a salt-free basis (77%). showed the following characterization: alpha cellulose, Anal. Ash as NazSOa: 21.0. Calcd. as NaCl, 17.3. 99.2%; ash, 0.040/o; iron, 10 p. p. m.; moisture, 4.0%. S (NaC1-free basis): 21.6. Calcd. for C6HlsOa(SGHs)2. The material was analyzed for pectin by the Link'J modi- 22.4. fication of the Lef&vre-Tollensloprocedure and was found A portion (1.5 9.) of the above salt-containing product to be pectin-free by this method. The cupranimonium was acetylated overnight with acetic anhydride (10 cc.) viscosity of the cellulose was determined in 0.1% concenand pyridine (5 cc.) and poured into 400 cc. of ice and tration a t 25 * 0.02': n = 2.732 centipoises; [ T ] (intrinsic water. A crystalline product was obtained that was char. 409.5 acterized as glucose diethyl mercaptal pentaacetate bv its In. __ 192.3 h?, melting point of 45-47" (mixed tti. p. unchanged); [ Q ] * ~ D viscosity) = = 7.559." C 0.1 +12.8" (c, 3.6; CHC13); yield, 2.1 g. (calcd., 2.17 g , ) . Reparation and Measurement of Cellulose Solutions.Wolfroml* recorded for this suhstance the constants: rn. p. Fuming hydrochloric acid (98.74 cc.; d*+ 1.203) was added 45-47''; [a]"D +11" (CHCl,). to the cotton linters (1.000 g.; moisture content, 4.0Oj,) Mercaptalation of Cellulose Hydrolytic h.oducts.-AIl i n a glass-stoppered flask and the mixture shaken until the amount of 30 g. of cellulose was dissolved in 600 cc. of cellulose was completely dissolved (eight minutes) with fuming hydrochloric acid (d164 1.203) by shaking in a 1occasional cooling in a n ice-bath. The density of the so- liter glass-stoppered bottle. After seventeen minutes a clear solution (dl64 1.219) was obtained that contained (8) Furnished through the courtesy of the Hercules Powder Co., 4.669 g. of cellulose per 100 cc. of solution. This solution Hopewell, Va. (9) A. D. Dickson, H. Otterrori and K . P. Link, THISJOURNAL, was kept in a thermostated constant temperature bath 6.3, 776 (1930). held at 16 * 0.1". The water in Lhe bath was cooled by (10)K . U. Lelkvre m d B . 'lollens, Ber , 40, 4618 (19071 ~iiedrisof ail iirir~ierseclC O ~ I which was connected with a (11) E. 0 Krnemer and W . 1) I.ansitig, J Phvs Chew., 39, 157 .. . .- .._..-. E
_.
-
~
(1936)
(12) M . I . . Woifrwl, 'IHLX
J O I J I ~ N A I . 61, ,
2188 (1929).
286
M. L. WOLFROM AND LOUISW. GEORGES
refrigeration unit. Samples of the soiution under investigation were removed at stated intenrals for mercaptala tion. Polarimetric readings at 16' were taken simultaneously and when plotted showed again a continuous curve. The mercaptalation products were isolated and analyzed. In the experiments herein reported, no attempts were made to isolate the various mercaptalated oligosaccharides in pure form. The data are tabulated in Table 11. The various mercaptalations are denoted by letter in this table and will be referred to by these designatiions. The mercaptalation procedure employed was that described for d-glucose, unless otherwise specified. An amount of 60 cc. (equivalent t o 2.80 g. of cellulose) of the solution was transferred to a glass-stoppered pressure bottle, 5 cc. of water and 8 cc. of purified ethyl mercaptan were added and the whole shaken mechanically for fifteen minutes a t 0". The addition of a small amount of water at this point lowered the acid concentration to that of ordinary concentrated hydrochloric acid. The product was precipitated by pouring the solution gradually into a suspension of 68 g. of sodium bicarbonate in 200 cc. of water. The excess bicarbonate was neutralized with acetic acid. The material was centrifuged and the residue washed successively with water, ethanol, acetone and petroleum ether. The washed product was dried in a vacuum oven a t 50". The mother liquors from the precipitates were tested with Fehling's solution and negative results were obtained in each case. They were then concentrated at 55", under reduced pressure to a moist salt and this transferred to a Biichner funnel, washed with acetone and dried at 40'. The salt residue was then acetylated for two days a t room temperature (occasional shaking) with acetic anhydride (38 cc.), and pyridine (18 cc.) and the acetylation product isolated by pouring into ice and water (800 cc.). The data obtained are tabulated in Table 11. The data tabulated in Table I1 were calculated to their average degrees of polymerization by means of the sulfur analyses and these calculated results are shown in Table 111.
VOl. 59
SUmmarY 1. Ethyl mercaptan has been used as a reagent to follow the course of hydrolysis of the cellulose molecule in fuming hydrochloric acid. 2. The hydrolysis of cellulose (chemically purified linters of high viscosity) in fuming hydrochloric acid at 23' was followed polarimetricaIly and by viscosity measurements. The rate of increase of the optical rotation from 0' to that of 100') was found to be a cond-glucose ([&ID tinuous function. The calculated monomolecular velocity constants showed a slight but definite upward drift. At an early stage ([a],, 25', approx.) of the hydrolysis the viscosity of the solution approached that of a solution containing an equivalent amount of apparent dglucose. 3. The course of hydrolysis of cellulose in fuming hydrochloric acid solutions (5%) a t 16' was studied by means of the mercaptalated products obtained from the reaction of ethyl mercaptan with the cellulose solutions a t various stages of hydrolysis. Simultaneous polarimetric data were obtained. Control experiments were made to determine the optimum procedure for the mercaptalation of d-glucose and this procedure was used in the cellulose reactions. Sulfur analyses served to determine the average molecular size of the mixtures of mercaptalated products isolated from the cellulose hydrolysis. 4. The data indicate a rapid hydrolysis to surprisingly small units. When the hydrolysis was approximately a t the half-period, the isolated The authors are indebted to E. I. du Pont products corresponded to the trisaccharide stage. de Nemours and Co., Inc., for the fellowship Glucose diethyl mercaptal first appeared when grant which has made this research possible, and the hydrolysis was approximately two-thirds to Dr. Robert W. Rothrock for practical assist- completed and gradually increased in amount. COLUMBUS, OHIO RECEIVED OCTOBER6, 1936 ance in the experimental work.
+
+
