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An investigation of the efficiency of dimethyl sulphate as a methylating agent for carbohydrates Glennie, Douglas William 1951

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AN INVESTIGATION OP THE EFFICIENCY OF DIMETHYL SULPHATE AS A METHYLATING AGENT FOR CARBOHYDRATES by DOUGLAS WILLIAM GLENNIE A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of Chemistry We aceept t h i s thesis as conforming to the standard required from candidates for. the degree of MASTER OF ARTS Members of the Department of Chemistry THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1951 ABSTRACT Dimethyl sulphate has been used for carbohydrate raethylation under various conditions of concentration, time and alkalinity i n order to determine optimum reaction conditions* Mesquite gum was used as a representative water-soluble polysaccharide. ACKNOWLEDGMENT The author i s indebted to Dr. E.V. White, who suggested this thesis problem and offered valued assistance and guidance throughout the course of work. - i i i -TABLE OP CONTENTS PAGE INTRODUCTION 1 PURPOSE OP STUDY 2 LITERATURE SURVEY . 2 PLAN OP INVESTIGATION . . . 5 EXPERIMENTAL METHODS AND RESULTS • • 8 Part 1. P u r i f i c a t i o n of crude mesquite gum . . . . 8 Part 2. Comparison of continuous methylation and stepwise methylation e f f i c i e n c y 10 Part 3. Minimum time of methylation f o r equi-valent substitution under constant conditions . . . 11 Part 4. Relation between the e f f i c i e n c y of methylation and t o t a l reagent concentration; the methylation e f f i c i e n c y and a l k a l i concentration . . 14 Part 5. The ef f e c t of methyl alcohol and dimethyl ether on the methylation e f f i c i e n c y • 17 Part 6. Rate of hydrolysis of dimethyl sulphate i n aqueous and al k a l i n e media; heat of reaction during alkaline hydrolysis of dimethyl sulphate and during methylation. 17 Part 7. S p e c i f i c r o t a t i o n measurements on p a r t i a l l y methylated mesquite gum samples . . . . . 24 DISCUSSION . . . . . . 25 SUMMARY AND CONCLUSIONS 31 i v BIBLIOGRAPHY APPENDIX • j LIST OP TABLES ' ' ; " PAGE Table 1. A n a l y t i c a l values f o r mesquite gum preparations 10 Table 2. Summary of methylation series 1, 2 and 3 (Part 2) • • • 1 2 Table 3> E f f i c i e n c y of reagent i n methylation series 1, 2 and 3 (Part 2) 13 Table 4. Extent of methoxyl sub s t i t u t i o n with decrease i n reaction time under constant methylation conditions (series 1 and 2) . . . . . . . . . . . • 14 Table 5, Summary of r e s u l t s i n Part 4 showing reagent r a t i o s and methoxyl content of p a r t i a l l y methylated products . . . . 15 Table 6. Calculated reaction e f f i c i e n c i e s (Part4), • 15 Table 7. Results of methylation with 2, 5, 11,1, 15.5 and 20$ sodium hydroxide (series l ) . Results at very low a l k a l i concentration (series 2). Results on the e f f e c t of methyl alcohol and d i e t h y l ether (series 3) 18 Table 8. Aqueous and a l k a l i n e hydrolysis data for dimethyl sulphate . . . . . . . . . . . . . . . . • 19 Tables 9-18. Temperature r i s e during hydrolysis of dimethyl sulphate with sodium hydroxide, ammonium hydroxide and during methylation 21 Table 19. S p e c i f i c rotation of p a r t i a l l y methylated mesquite gum products and methoxyl content . . . . . 24 -v-LIST OF FIGURES Fig* 1. Variation in methoxyl content with time of methylation for series 1, 2 and 3. Fig. 2. Average efficiency and efficiency for each reaction with progressive methylation. Fig. 3. Methoxyl content of partially methylated products with time of reaction under constant methylation conditions. Fig. 4. Variation in methoxyl content and efficiency with total reagent concentration; comparison with series 3, Part 2. Fig. 5. Methoxyl content and efficiency versus alkali concentration. Fig. 6. Rate of hydrolysis of dimethyl sulphate in water, alkali and salt solutions at various temperatures. Fig. 7. Temperature variation during the alkaline hy-drolysis of dimethyl sulphate. Fig. 8. Comparison of the variation in temperature during hydrolysis and methylation. Fig. 9. Specific rotation of partially methylated products versus methoxyl content. - v i -AN INVESTIGATION OF THE EFFIGIENGY OF DIMETHYL SULPHATE AS A METHYLATING AGENT FOR CARBOHYDRATES by DOUGLAS WILLIAM GLENNIE INTRODUCTION The methylation of carbohydrates i s undoubtedly one ©f the most important reactions i n the f i e l d of carbo-hydrate chemistry. It i s used extensively i n determining the structure of n a t u r a l l y occurring carbohydrate substances as well as those prepared s y n t h e t i c a l l y . In the words of B e l l ( l ) : " I t i s perhaps i n t e r e s t i n g to r e c a l l that, despite so many advances i n technique, methylation, o r i g i n a l l y conceived i n the St. Andrews laboratory of Purdie and Irvine, remains an es s e n t i a l t o o l f o r a l l fundamental s t r u c t u r a l investigations". As a research t o o l the methylation reaction plays an indispensable part i n studies on the nature of linkages between component uni t s of a carbohydrate compound. During methylation free hydroxyl groups not involved i n such linkages are e t h e r i f i e d . Under mild hydrolysis these linkages may be s p l i t to y i e l d the i n d i v i d u a l component units containing a free hydroxyl group only wherever linkage existed. This method of " l a b e l l i n g " free hydroxyl groups by forming t h e i r methyl ether derivatives finds some analogy i n the use of traeer technique i n radiochemistry. This same -a- . procedure also forms the basis f o r the so-called "end group" method of determining chain length i n long chain poly-saccharides (2,3). Studies of carbohydrate methylation have afforded some information on the r e l a t i v e r e a c t i v i t y of constituent hydroxyl groups(4-9). F i n a l l y , the methylation reaction has been employed i n industry f o r the preparation of methyl derivatives such aB methylcellulose which f i n d use i n the manufacture of films and plastics(10-13)• PURPOSE OF STUDY Dimethyl sulphate and a l k a l i , as a methylating agent, has received extensive use i n carbohydrate studies. In the methylation of carbohydrates, however, i t i s often necessary to repeat the methylation treatment several times before maximum subs t i t u t i o n i s achieved. Even a f t e r repeated methylation i n some cases s u b s t i t u t i o n i s incomplete. Despite many modifications of the reaction conditions, there i s apparently no data available on the e f f i c i e n c y of reaction at various degrees of substitution or on the r e l a t i v e e f f i c i e n c y of reaction under varied conditions. These problems were investigated i n an e f f o r t to f i n d optimum methylation conditions. LITERATURE SURVEY , The f i r s t method f o r e t a e r i f y i n g sugars,was introduced by Purdie and Irvine(14), who employed, methyl iodide and s i l v e r oxide as a methylating agent. In t h i s method reducing sugars are converted to their,glycosides •2-before methylation i n order to prevent oxidation by the s i l v e r oxide reagent. In the presence of solvents such as methyl alcohol side reactions (formation of methyl ether) reduce the reaction e f f i c i e n c y considerably. However, as the degree of sub s t i t u t i o n increases the s o l u b i l i t y i n methyl iodide increases and complete substitution i s f a c i l i t a t e d . For t h i s reason "Purdie's reagents" are found to be most e f f e c t i v e i n advanced stages of methyl-a t i o n (15-18). Denham and Woodhouse(l9) introduced dimethyl sulphate and a l k a l i to the carbohydrate f i e l d i n t h e i r studies on the methylation of c e l l u l o s e . Haworth(20) l a t e r extended the use of these reagents to the methylation of simple sugars. Methylation was carr i e d out by dropwise addition o f dimethyl sulphate and 30$ sodium hydroxide to a concentrated aqueous solution of the sugar kept vigorously s t i r r e d . The rate of addition was adjusted i n such a manner as to maintain the reaction mixture s l i g h t l y a l k a l i n e as a precaution against hydrolysis. The heterogeneous nature of the reaction mixture necessitates vigorous s t i r r i n g and side reactions necessitate the use of a large excess of reagents. Owing to the competitive nature of the reaction, the e f f i c i e n c y i s low. Nevertheless, the Haworth process i s widely used, e s p e c i a l l y f o r preliminary methylation (15-18,20-22). Many modifications of the o r i g i n a l Haworth procedure have been made i n connection with studies on the methylation of c e l l u l o s e . One of these, developed i n -3-the Haworth laboratory(23), consists of a simultaneous regeneration and methylation treatment of ce l l u l o s e acetate dissolved i n acetone. I t was found that there was no advantage gained by substituting 45% potassium hydroxide f o r the usual 50% sodium hydroxide. Lithium hydroxide gave i n f e r i o r y i e l d s with a low degree of sub-s t i t u t i o n . I t has been shown(24) that sodium methyl sulphate with sodium hydroxide i s e f f e c t i v e i n the prepara-t i o n of low but uniformly substituted methylcellulose. In a l l these modifications, which have been reviewed(lO), the physical c h a r a c t e r i s t i c s of c e l l u l o s e play a dominant r o l e and the methods are not of general a p p l i c a b i l i t y . Haworth and co-workers have also applied the simultaneous regeneration-methylation technique to the methylation o f starch and glycogen(25,26). The methylation of starch with dimethyl sulphate and other a l k a l i e s , such as ammonium hydroxide, has been suggested(27). In some instances carbohydrates containing resis t a n t hydroxyl groups, p a r t i c u l a r l y primary hydroxyl groups(22), have been methylated with m e t a l l i c sodium, potassium or l i t h i u m i n l i q u i d ammonia and other solvents (21,22,28-32). The e f f i c i e n c y of the reaction i s l i m i t e d by the low s o l u b i l i t y of the a l k a l i metal derivatives(29) and there i s some controversy as to whether concurrent decomposition r e s u l t s i n t h i s reaction(31-33). Qther methods of carboydrate methylation are recorded i n the l i t e r a t u r e . These methods are usually ^slight modifications of the Purdie or Haworth methods or involve, i n some cases by necessity(15,18), a combination of these treatments. The important ones, from a s t r u c t u r a l standpoint, are reviewed by B e l l ( l ) . PLAN OF INVESTIGATION To i n i t i a t e t h i s study a carbohydrate suitable for methylation was required. Mesquite gum, a water soluble gum somewhat res i s t a n t to methylation, was readily available and judged to be representative. The structure of the gum, as proposed by White(34-37), i s shown diagramatically. Other studies by Cuneen and Smith(38) indicate that the structure i s more complex than that represented. For s i m p l i c i t y the structure i n t h i s work i s taken to be the free acid hepta-saccharide of molecular weight 1061 containing one methoxyl group and 17 hydroxyl groups'available f o r methylation. -D-GALACTOSE- JL46. -D*-GALACTOSE-I 1*3 I 4^Me0-D-GLTJCURONIC AGID ( s a l t , ester) 1:2 L-ARABINOSE l i s L-ARABINOSE 1:2 L-ARABINGSE •ItS L-ARABINOSE n To prepare the crude mesquite gum at hand for characterization and methylation studies a preliminary i n v e s t i g a t i o n on a method of p u r i f i c a t i o n was i n order. In t h i s connection the methods of White(34) and Anderson *and Otis(39) were ref e r r e d to. I t was decided that no -5-e f f o r t should be made to prepare the free acid gum by repeated treatment with mineral a c i d , since there i s a danger of hydrolysing the l a b i l e pentose u n i t s . Results expressed on the ashless basis were assumed to take into account any v a r i a t i o n i n the degree of s a l t formation i n the gum. In order to assess the e f f i c i e n c y of methylation reactions a method of analysis was required. I t was decided that the percentage dimethyl sulphate actually u t i l i z e d for methylation could be taken as a c r i t e r i o n of e f f i c i e n c y . I t was seen that, with t h i s c r i t e r i o n and a knowledge of the y i e l d of methylated product, the i n i t i a l and f i n a l methoxyl content, and the amount of dimethyl sulphate used for reaction, an estimation of the reaction e f f i c i e n c y could be made. With t h i s i n mind the techniques of methoxyl analysis were practised on standard v a n i l l i n samples u n t i l reproducible accuracy was obtained. A l l methoxyl analyses were c a r r i e d out by the method of Viebock and Schwappach(40) as modified by Glark(41). The purpose of the study has already been out-l i n e d . However, s p e c i f i c problems deemed worthy of consideration may be mentioned. One of these i s the problem of continuous methylation versus stepwise methylation. In p a r t i c u l a r , a comparison of the e f f i c i e n c y of repeated methylation with the e f f i c i e n c y of methylation i n one continuous operation was desired. I t was also desired to know the minimum time of methylation for equivalent sub-s t i t u t i o n or time a f t e r which no further substitution occurs. -6-Another problem a r i s e s i n consideration of the methylation e f f i c i e n c y as the t o t a l reagent concentration varies* Closely connected to t h i s problem i s one concerning the methylation e f f i c i e n c y with v a r i a t i o n i n a l k a l i concentra-t i o n alone* Linked with the question o f v a r i a t i o n i n t o t a l reagent concentration and a l k a l i concentration i s the question of eff e c t of d i f f e r e n t solvents on the methylation e f f i c i e n c y . S t i l l another pertinent problem i s to know something of the extent of hydrolysis or saponi f i c a t i o n of dimethyl sulphate under methylating conditions i n the presence and absence of carbohydrate material.. Although a knowledge of the temperature dependence of methylation e f f i c i e n c y i s important, time did not permit i n v e s t i g a t i o n of t h i s phase. On the other hand, some insight into the problem may be gained through a knowledge of the heat of reaction during methylation. F i n a l l y , i t i s of some intere s t to know the r e l a t i o n , i f any, which ex i s t s between the s p e c i f i c r o t a t i o n of p a r t i a l l y methylated products and t h e i r methoxyl content. I t may be seen that, i f a well defined r e l a t i o n e x i s t s , the s p e c i f i c r o t a t i o n measurements would be a convenient supplement to methoxyl analyses. The foregoing plan i s intended only as a summary to the experiments described i n d e t a i l i n the next section and the problems mentioned are discussed more f u l l y under the "Discussion". EXPERIMENTAL METHODS AND RESULTS For convenience description of the techniques and methods employed throughout the investigations enumerated i n the previous section i s grouped under seven headings. These headings are l i s t e d below: Part 1. P u r i f i c a t i o n of crude mesquite gum. Part 2, Comparison of continuous methylation and stepwise methylation e f f i c i e n c y . Part 3, Minimum time.of methylation for equivalent substi-t u t i o n under constant conditions. Part 4, Relation between the e f f i c i e n c y of methylation and t o t a l reagent concentration; the methylation e f f i c i e n c y and a l k a l i concentration, , ,* ... Part 5, The e f f e c t of methyl alcohol and dimethyl ether on the methylation e f f i c i e n c y , „,. > ] ?, ! ; 3. n». Part,, 6,. Rate of hydrolysis of dimethyl sulphate i n aqueous and a l k a l i n e media; heat of reaction during al k a l i n e hydrolysis of dimethyl sulphate and during methylation. Part 7, S p e c i f i c r o t a t i o n measurements on p a r t i a l l y methylated mesquite gum samples. Part 1, P u r i f i c a t i o n of crude mesquite gum, A 25 g, sample of crude mesquite gum was placed i n a beaker with 50 ml. of d i s t i l l e d water and allowed to stand overnight. The syrup obtained was s t i r r e d with an addi t i o n a l 200 ml, of water to y i e l d a t h i n syrup of -8-approximately 9% solution. This t h i n syrup was f i l t e r e d through kieselguhr to remove bark p a r t i c l e s and other impurities. The f i l t r a t e , a pale yellow t h i n syrup, was dialyzed i n cellophane against cold tap water for 24 hours to y i e l d a f i n a l volume of 330 ml. This volume was reduced to 130 ml. by evaporation under reduced pressure at 30°. An equal volume of 95% ethanol was added and the soluti o n poured i n a f i n e stream into 1500 ml. of 95% ethanol made B/5 with sulphuric acid. The milky suspension obtained was allowed to s e t t l e and was f i l t e r e d and washed with 95% ethanol, dry ethyl ether, and f i n a l l y with l i g h t petroleum ether. The product was pulverized and dried to y i e l d a white amorphous powder, r e a d i l y soluble i n water, aci d and d i l u t e a l k a l i but insoluble i n organic solvents; y i e l d 20 g. A second preparation from 100 g. of crude mesquite yielded 85 g. of p u r i f i e d product. In a t h i r d preparation 300 g. of crude gum were dissolved i n water, f i l t e r e d and s t i r r e d with 60 ml. of concentrated hydrochloric acid for one h a l f hour. A f t e r d i a l y s i s the p u r i f i e d product was i s o l a t e d and dried as before; y i e l d 210 g. A summary of the y i e l d s and a n a l y t i c a l values fo r each product, numbered i n order of preparation, i s given i n Table 1. Included i n the table are values f o r mesquite gum published by Otis and Anderson(38) and values calculated from the structure of mesquite gum proposed by White(34-37). The calculated values are based on the structure i l l u s t r a t e d on page 5. Table 1. A n a l y t i c a l values f o r mesquite gum preparations. Value Sample 1 Sample 2 Sample 3 (38) (.34-37) % Recovery 81.0 85.0 70.0 Methoxyl 2.43 2.40 2.44 2.86 2.92 Pentosan 47.02 56.6 Carbon dioxide 3.86 3.55 4.15 Carbon 45*46 44.10 Hydrogen 6.39 6.08 Ash 2.14 2*52 0.85 0.07 0.00 Mol. wt. 1222 1061 c°or 68.3' 70.8° Part 2. Comparison of continuous methylation with stepwise methylation e f f i c i e n c y . In this, i n v e s t i g a t i o n 5 g. samples of p u r i f i e d mesquite gum were used throughout. Samples were dissolved i n the required amount of d i s t i l l e d water i n stoppered fl a s k s and shaken with dimethyl sulphate and 30$ sodium hydroxide on a mechanical shaker f o r i n t e r v a l s of one hour. The temperature of methylation was maintained over the range 20-25° by cooling when necessary. P a r t i a l l y methylated gum solutions were dialyzed to remove spent reagents and evaporated to dryness under reduced pressure at 30°. Samples of approximately 3 g. were removed from re a c t i o n vessels and dried f o r analysis. In the f i r s t methylation series samples of p u r i f i e d gum were methylated for one, two, three and four hours with d i r e c t addition of reagents to the reaction mixtures at the end of -10-each one hour period. In a second methylation series step-wise methylation was c a r r i e d out on samples for one, two, three, and four hour periods. Reaction mixtures were d i a -lyzed, evaporated to dryness and the methylation treatment repeated once, twice and t h r i c e . In a t h i r d methylation s e r i e s samples were methylated continuously i n the same manner described f o r the f i r s t series except time i n t e r v a l s of 15 minutes were used throughout. The r e s u l t s are summarized i n Table 2. In Pig. 1. a p l o t of methoxyl content with time of methylation f o r each series i s shown. Where duplicate runs were made the average methoxyl values are graphed. F i g . 2 i l l u s t r a t e s the v a r i a t i o n of methylation e f f i c i e n c y with number of methylations f o r each seri e s . Sample calculations of reaction e f f i c i e n c y are shown i n the Appendix, page i and the e f f i c i e n c y values are l i s t e d i n Table 3. BarjL-£. Minimum time of methylation for equivalent s u b s t i -t u t i o n under constant conditions. A study was made of the extent of su b s t i t u t i o n effected under constant methylating conditions when the reaction time i s systematically reduced. In the f i r s t s e r i e s , series 1, 5 g, samples of p u r i f i e d gum were dissolved i n 10 ml. of d i s t i l l e d water and shaken with 5.0 ml. of dimethyl sulphate and 5.83 ml. of 30$ sodium hydroxide i n stoppered f l a s k s on a mechanical shaker. A f t e r shaking for the desired time reaction mixtures were dialyzed immediately and the p a r t i a l l y methylated gum products i s o l a t e d by evaporation and drying as described i n Part 1. In a second s e r i e s , -11-Table 2. Summary of methylation series 1, 2 and 3. 30$ NaOH Series & sroduct Starting material Water Dimethyl sulphate 1-1 1-2 1-3 1-4 1-5 1-6 1- 7 2- 1 2-2 2-3 2- 4 3- 1 3-2 3-3 3-4 Sample 1 n Sample 2 tt Sample 2 Sample 3 Sample 3 10 ml, it ii M 1© 10 ml) • J 10 ml. II 10 ml. 5,0 ml, it 5,0 ml, ti 5,83 ml. - i a -Yield % Yield MeO % Ash %MeO (Ashless) 4,5 g. 4,85 3,80 90 97 76 14.0 15.8 23.3 2.66 4.08 2.65 14.4 16.5 24.0 5,0 100 23.0 2.75 23.6 5.0 100 28.7 2.04 29.3 4,6 92 29.6 2.11 30.2 4,5 90 30.3 2. 36 31.1 5,0 100 25.5 2.16 26.1 5.0 100 26.5 2.32 27.1 4*2 84 32.8 2.62 33.7 4.5 4-5 g. 90 90 32.3 16.6 2. 35 1.91 33.1 16.9 4-5 90 24.4 1.92 24.9 4-5 90 28.7 2.06 29.3 4-5 90 29.3 1.98 29.9 Table 3. Efficiency of reagent,methylation series 1, 2 and 3. Series and Product ~ 1st methylat ion " 2nd methylat Lon 3rd methylati on 4th methylation Overall methylation MeO gps» introd. E f f i c . MeO gps. introd. E f f i c . MeO gps. introd. E f f i c . MeO gps. introd. E f f i c . MeO gps. introd. E f f i c . 1-1,2 1-3,4 1-5,6 1-7 2- 1,2 2- 3,4 3- 1 3-2 3-3 3-4 4*60 II II ii ii . .  it 5.16 ii it ii 41.0 n II ii II . -it 46.1 it ii ii 3.4 it M 4.6 ti 3.32 II n 30.4 it it 41.0 ti 29.6 ti it 2.60 it 3.10 1.92 it 23.2 II 27.6 CM 17.1 it 1.6 mm 0.3 5.35 2.68 4.60 8.0 10.6 11.2 ~ 9.2 12.3 5.16 8.48 10.4 10.7 41.0 35.7 31.5 25.0 41.0 36.8 46.1 37.8 30.9 23.9 -13-Figure-1» V a r i a t i o n i n methoxyl content (ashless) with time of methylation for series 1, 2 and 3. M a x i m u m fsAeO (calculated) series I series 3 NaOH _ t i 0 MeiS04~ M e , S 0 4 = 1 ' i Z Gum i 3 A-Number of methyl at ion 5 Figure 2, Average e f f i c i e n c y and e f f i c i e n c y f o r each reaction with progressive methylation, Number o*f metfty/oti'ons s e r i e s 2, samples were methylated i n the same manner but reaction mixtures were neutralized with hydrochloric acid before d i a l y s i s . The re s u l t s are shown i n Table 4 and graphically i n Pig, 3. Tab_le_4, The extent of methoxyl sub s t i t u t i o n with decrease i n reaction time under constant methylation conditions (series 1 & 2), Series and Product Sta r t i n g material Time Y i e l d Y i e l d ' % MeO Ash % Methoxyl (ashless! 1-1 Sample 2 30 m. 4.5 g. 90 15.9 2.56 16.3 1-2 ii 15 4-5 80-90 16.0 2.01 16.3 1-3 H 7.5 4-5 80-90 15.5 2.23 15.9 1-4 ti 3.75 4-5 80-90 15.7 2.09 16.0 2-1 Sample 3 15. m. 4.5 g. 90 16.3 1.50 16.6 2-2 ii 7.5 4-5 80-90 16.5 1.93 16.8 2-3 it 3.75 4-5 80-90 13.6 1.87 13.9 Part 4. Relation between the e f f i c i e n c y of methylation and t o t a l reagent concentration; the methylation e f f i c i e n c y and a l k a l i concentration. For the purpose of the f i r s t study, a number of samples of p u r i f i e d gum were methylated i n the same manner described i n Part 1 and Part 2 with the exception that, while the r a t i o of 30$ sodium Hydroxide to dimethyl sulphate was maintained at 1.1, the r a t i o of dimethyl sulphate and a l k a l i to gum was varied. In each case a 5 g. sample of p u r i f i e d gum (sample 3) was dissolved i n 10 ml. of d i s t i l l e d water and methylated f o r 15 minutes and the reaction mixture imme-di a t e l y dialyzed. The f i n a l products were i s o l a t e d for analysis i n the usual manner. The res u l t s are summarized i n -JL4-Figure 3 . Methoxyl content of p a r t i a l l y methylated products with time of reaction under constant methylation conditions. 25 V —c T i 8 10 ime in m -© o — series 1 0— series Z NjiPJi . i.io M e , S 0 » . 1.12 Irxvt J5 es 20 Table 5 and shown graphically i n Pig, 4. The curve from Series 3, Fig, 1 i s included i n Fig, 4 for comparison. Cal-culated efficiencies are li s t e d i n Table 6 and a plot of the efficiencies i s also shown i n Fig, 4, The curve from Series 3, Fig. 2 of the overall efficiency is again included for comparison. Table 5, Summary of results i n Part 4 showing reagent ratios and methoxyl content of partially methylated products. Product KaOH Me£0 4 MeSOi Gum ^ — Yield % ~ MeO Ash % MeO (ashless) 1 11.7 ml. 10 ml. 2.24 90 23.6 1.92 24.1 2 17.5 15.0 3.36 90 26.1 2.08 26,7 3 23.3 20.0 4.48 90 27.1 1.71 27.6 Table 6. Calculated reaction efficiencies (Part 4). Product MeSO. Gum Me£0* avail. OH MeO gps. introduced Efficienegr 3-l(Pt.l) 1.12 0.70 5.16 46*1 % 1 2.24 1.40 8.13 36.3 2 3. 36 2.10 9.20 27.3 3 4.48 2.80 9.60 21.4 For the purpose of the second study, samples of purified mesquite gum were methylated i n the usual manner but the alk a l i concentration was varied in an effort to determine the optimum concentration with^respect to time of reaction, extent of substitution and reagent- efficiency. In a series of experiments 5 g. samples of gum were dissolved i n d i s t i l l e d water, a l k a l i added and the solution shaken in stoppered flasks with 5 ml, of dimethyl sulphate for 15 -15-Figure 4. V a r i a t i o n i n methoxyl content and e f f i c i e n c y with t o t a l reagent concentration and comparison between series 3, Part 2. \ I I I L. Raiio of Me*50 4 -to gum minutes. In every ease the molar ratio of sodium hydroxide to dimethyl sulphate was kept constant at 1.1 Results with 2,5,11.1,15.5, and 20$ sodium hydroxide are li s t e d i n Table 7. In two separate experiments the methylation e f f i -ciency was investigated at very low alkal i concentrations. In the f i r s t experiment a 5 g. sample of purified gum was dissolved i n 10 ml. of d i s t i l l e d water and shaken with 5 ml. of dimethyl sulphate. A total of 5.83 ml.(10$ excess) of 30$ sodium hydroxide was added dropwise (about 1 drop per minute) such as to maintain the pH between 7 and 9. The reaction required 2.5 hours for completion as evidenced by constant pH of the reaction solution. Results from this experiment are included i n Table 7 under experiment 2-1. In the second experiment a 5 g. sample of puri-fied gum was dissolved i n a buffer solution consisting of 30 ml. of d i s t i l l e d water containing 20.8 g. of Na^HP04.12H20. During the reaction 0.3 ml..of 30$ sodium hydroxide was added dropwise over a total shaking period of 45 minutes such as to maintain the pH between 7 and 8. The product (2-2) was iso-lated for analysis i n the usual manner and results are l i s t e d i n Table 7. In a third separate experiment the effect of re-placing sodium hydroxide with ammonium hydroxide on the re-action efficiency was investigated. A 5 g. sample of puri-fied gum was dissolved i n 10 ml. of d i s t i l l e d water and shaken with 5.83 ml. of 18.8$ ammonium hydroxide (equivalent to 5.83 ml. of 30$ sodium hydroxide) and 5 ml. of dimethyl sulphate. The product (2-3) was isolated for analysis as -16-before and results are l i s t e d i n Table 7, Part 5. The effeet of methyl alcohol and diethyl ether on the methylation efficiency. In this investigation the reagent efficiency with water as solvent was compared with the efficiency with methyl alcohol-water (1:3 by volume) and diethyl ether-water (1:3) as solvent. In the f i r s t experiment 5 g, of purified gum were dissolved i n 15 ml, of methyl alcohol-water (1:2) and the solution shaken in the usual manner with 5 ml, of d i -methyl sulphate for 15 minutes. The second experiment was a repetition of the f i r s t with 15 ml, of diethyl ether-water (1:3) replacing the methyl alcohol-water solvent. Results are included i n Table 7 under product 3-1 and 3-2respectively, Part 6, Rate of hydrolysis of dimethyl sulphate i n aqueous and alkaline media; heat of reaction during alkaline hydro-l y s i s of dimethyl sulphate and during methylation. To study the hydrolysis of dimethyl sulphate 5 ml, portions of dimethyl sulphate were shaken with d i s t i l l e d water and also with sodium hydroxide solution for various time intervals i n the same manner i n which methylations were carried out. Aqueous hydrolysis was studied by shaking 40$ aqueous solutions of dimethyl sulphate for a given time and ti t r a t i n g the acidity with standard sodium hydroxide solution. In two series of alkaline hydrolysis dimethyl sulphate was shaken with 2$ and 11,1$ sodium hydroxide solution (10$ i n excess of Me SO^  for given periods and unneutralized sodium Table 7. -Results of methylation with 2,5,11.1,15.5, and 20$ sodium hydroxide (series l ) . Results at very low a l k a l i concentration (series 2). Results on the effect of methyl alcohol and diethyl ether (series 3). Series & Product Water 30$ NaOH MetS0A Time $ Yield $ MeO $ Ash $ MeO (ashless) MeO gps. introd. E f f i c . 1-1 81.8 ml. 5.83 ml. 5 ml. 15 min. 80-90 10.0 1.94 10.2 2.60 24. 6$ 29.3 10.0 5.5 P~ 9 ti II n it 13.1 1.88 13.4 3.82 36.2 1-3 1-4 i —^ it « tt tt 16.6 1.91 16.9 5.16 46.1 ii ii ti ti 18.3 1.92 18.7 5.89 55.7 ii ti ti ti 19.6 2.01 20.0 6.41 60.2 A —O 2-1 10.0 ti ft 25 hrso ii 18.4 1.87 18.8 5.94 56.2 2-2 u (dropwise) dropwise) ft 45 min. ii 2.6 2.04 2.66 -0.1 0 (buffer ) 2.82 -0.04 2-3 tt 5.83 ti 15 min. tt 2.80 0.64 0 3-1 3-2 MeOH-Et,0-(NHA0H ) 5.83 n ti it ti it it tt 12.6 18.8 1.91 1.93 13.0 19.2 3.67 6.10 34.7 57.7 -18-hydroxide t i t r a t e d with standard hydrochloric acid. During a l k a l i n e hydrolysis heat of reaction was allowed to proceed without compensatory cooling. During aqueous hydrolysis the temperature of the reaction mixture remained constant at the I n i t i a l temperature of 24°. Results are shown i n Table 8 and are plo t t e d i n Pig. 6. Dotted curves i n Pig. 6, taken from the work of Lewis, Mason and Morgan(42), are included f o r comparison. Table~8. Aqueous and alkaline hydrolysis data f o r ;dimethyl sulphate. D i s t i l l e d water 30 % NaOH Net [NaOH] Time % Meg>S,Q reaetea 10 ml. Ormn. 0.32 ti — — 5 0.90 it — - 10 1.10 it - - 15 1.73 II — — 20 2.04 ii _ _ 30 2.62 81.8 5.83 2 % 5 31.8 it it it 10 41.0 II it it 15 45.0 II II it 20 47.0 fi it ii 30 48.5 10.0 5.83 11.1 5 45.7 ii ti it 10 51.4 n it it 15 53.2 •i it ii 20 54.9 ii it ii 30 55.0 To study the v a r i a t i o n i n reaction heat during a l k a l i n e hydrolysis of dimethyl sulphate, 5 ml. portions of dimethyl sulphate were shaken with sodium hydroxide solutions f o r various time i n t e r v a l s i n the same manner i n which methyl-ations were performed, with the exception that the heat of reaction was not compensated f o r by cooling. At the end of each time i n t e r v a l the temperature of the reaction mixtures ( i n stoppered flasks) was recorded. In a l l cases the molar Figure 5. Methoxyl content and e f f i c i e n c y versus a l k a l i concentration. r a t i o of sodium hydroxide to dimethyl sulphate was maintained at 1,1. Results are l i s t e d i n Tables 9 to 18 and the various p l o t s of temperature with reaction time are shown i n Figs. 7 and 8. Pl o t s H and U 2 , F i g . 8, show the r i s e i n temperature when 5 g. of p u r i f i e d mesquite are methylated with 5 ml, of dimethyl sulphate and 11.1$ sodium hydroxide and the r i s e a f t e r further addition of an equal quantity of reagents. -20-Figure 6 . Rate of hydrolysis of dimethyl sulphate i n water, a l k a l i and s a l t solutions at various temperatures. 9o rf 80 J9£l MeiS04 (aqueous) i i - (95°) 2 # N o O H (24-34" ; z o 3 0 4 0 " T i m e i n m i n u t e s Table 9. Temperature change during hydrolysis of 5 ml. dimethyl sulphate with 87.6 ml. of 2% NaOH. Time Temp. Time Temp. Time Temp. 0 m. 1 2 3 4 5 24.0° 26.0 28.3 30.2 32.2 33.2 6 m. 7 8 9 10 11 33.8° 33.8 33.7 33.6 33.3 33.1 12 m. 13 14 15 20 32.8° 32.5 32.1 31.9 30.3 Table 10. Temperature change during hydrolysis of 5 ml. of dimethyl sulphate with 35.1 ml. of 5$ NaOH. Time Temp. Time Temp. Time Temp. 0 m. 25.0° 5 m. 44.8° 10 ra. 36.0° 1 29.0 6 43.8 15 31.0 2 33.0 7 40.7 20 28.4 3 39.3 8 39.2 — — 4 45.6 9 37.5 -Table 11. Tenroerature chance during hydrolysis of 5 ml. of dimethyl sulphate with 15.8 ml. of 11.1$ NaOH. Time Temp. Time Temp. Time Temp. 0 m. 24.0° 6 m. 49.0° 12 m. 34.4° 1 26.3 7 45.5 13 33.0 2 29.0 8 42.2 14 31.9 3 32.8 9 40.0 15 31.1 4 39.6 10 37.8 20 28.0 5 53.5 11 35.9 — — -21-Table 12. -Temperature change during hydrolysis of 5 ml, dimethyl sulphate with 11,3 ml. of 15$ NaOH, Time Temp, Time Temp. Time Temp. 0 m. 24,0° 6 m. 42.0° 12 m. 34. 3 c 1 24.5 7 56.2 13 32.3 2 26,0 8 49.5 14 30.8 3 27.9 9 44.3 15 29.5 4 31.0 10 40.1 20 26.0 5 35.0 11 36.9 -Table 13. Temperature change during hydrolysis of 5 ml. of dimethyl sulphate with 8.7 ml. of 20$ NaOH. Time Temp. Time Temp. Time Temp. 0 m. 25.0° 6 m. 29.8° 12 m. 46.0° 1 25.8 7 31.5 13 41.5 2 26.1 8 34.2 14 38.0 3 26.9 9 40.5 15 35.2 4 27.8 10 52.2 20 27.7 5 28.6 11 52.0 - — Table 14. Temperature change during hydrolysis of 5 ml. ©f dimethyl sulphate with 15.8 ml. of 18.8$ NH.OH Time Temp. Time Temp. Time Temp. 0 m. 25.0° 6 m. 43.3° 12 ra. 31. 0 C 1 71.0 7 40.2 13 29*9 2 66.0 8 37.6 14 29.1 3 58.2 9 35.5 15 28,6 4 52.2 10 33.6 20 26.5 5 47.4 11 32.1 — — ,-22-Table 15. Temperature change during hydrolysis of 5 ml. of dimethyl sulphate with 15.8 ml. of 11.1$ NaOH and 5 ml. of MeOH. Time Temp. Time Temp. Time Temp. 0 m. 1 24.0° 34 .0 1.5 2.0 55° -explosion Table 16. Temperature change during hydrolysis of 5 ml. of dimethyl sulphate with 15.8 ml. of 11.1$ NaOH and 5 ml. of ether. Time Temp. Time Temp. Time Temp. 0 m. 24.0° 4 31.3° 8 m. 35.2° 1 26.7 5 32*8 9 34.5 2 28.2 6 34»2 10 33.2 3 30.0 7 35.2 -Table 17. Temperature change during methylation of 5 g. gum with 5.0 ml. dimethyl sulphate 15.8 ml. of 11.1$ NaOH. Time Temp. Time Temp. Time Temp. 0 m. 25.0° 6 m. 46.0° 12 m. 33*6° 1 28.0 7 43.0 13 32.1 2 36.8 8 40.3 14 31.3 3 53.2 9 38.3 15 30.3 4 53.0 10 36.5 20 27.8 5 49.8 11 35.0 - -Table 18. Temperature change during 2nd methylation follow-ing addition of 5 ml. of Me2SG-4& 5.83 ml. of 30$ NaOH. Time Temp. Time Temp. Time Temp. 0 m. 25.0° 6 m. 41.3° 12 m. 33.5° 1 30.0 7 39.6 13 32.8 2 39.2 8 38.1 14 31.9 3 47.0 9 36.8 15 31.4 4 45.3 10 35.6 20 30.0 5 43.1 11 34.7 — — -23-Figure 7, Temperature v a r i a t i o n during a l k a l i n e hydrolysis of dimethyl sulphate. f l - NaOH IL- 51 » BT-J5.51 « 20^ Y - NM4OH S I - J I . l l NaOH ( M e O r O m - i l . l l N a O H T i 10 m e fn If /JO minutes IS Figure 8. Comparison of the v a r i a t i o n i n temperature during hydrolysis and methylation. Part 7. Specific rotation measurements on pa r t i a l l y methylated" mesquite gum samples. Partially methylated products prepared i n previous experiments were dissolved i n d i s t i l l e d water and the optical rotation measured i n an effort to determine any relation be-tween methoxyl content and specific rotation. It was nec-essary to employ dilute solutions i n order to obtain s u f f i -cient light penetration through the solutions. Samples were weighed directly into volumetric flasks and allowed to dis-solve slowly i n water overnight i n order to avoid froth formation which accompanies agitation of the solutions. He-suits are l i s t e d i n Table 19 and a plot of specific rotation against methoxyl content i s shown i n Fig, 9, Table 19* Specific rotation of partially methylated mesquite gum products and methoxyl content. Part-Series - Product Methoxyl content M : Part-Series - Product Methoxyl content 1-0-2 2,40 +59.3° 2-3-1 +16.9 +56.3 ° 1-0-3 2.46 68.3 4-1-1 24.1 58.4 6-1-1 10.2 56.2 2-3-2 24.9 51.0 ,6-1-2 13.4 55.4 2-2-1 26.1 42.4 3-2-3 13.9 57.0 4-1-3 27.6 58.8 3-1-3 15.9 37.5 2-3-3 29.3 50.5 3-1-4 16.0 56.3 2-3-4 29.9 57.8 2-2-2 16.5 57.7. 2-2-3 33.7 44.3 -24-Figure 9. S p e c i f i c r o t a t i o n of p a r t i a l l y methylated products vs. methoxyl content 'DISCUSSION The r e s u l t s of studies i n Part 2 show that methyl-ation i n steps i s more e f f i c i e n t than continuous methylation, p a r t i c u l a r l y i n the advanced stages of substitution. I t may be seen i n Pig, 1, that a f t e r a methoxyl content of about 25$ i s reached the rate/of s u b s t i t u t i o n for continuous methylation decreases and at about 30$ methoxyl content the rate i s almost zero. On the otiaer hand, the rate of stepwise su b s t i t u t i o n decreases to a l e s s e r extent and remains f i n i t e above a meth-oxyl content of 35$, An examination of the e f f i c i e n c i e s (Table 3) shows that a f t e r 2 additions of reagents the over-a l l e f f i c i e n c y f o r continuous re a c t i o n has dropped by 10 to 15$ while a f t e r 2 repeated reactions the e f f i c i e n c y has f a l l e n by only 4,2$, The res u l t s show also that as the degree of s u b s t i t u t i o n increases or the available hydroxyl concentra-t i o n decreases the rate of sub s t i t u t i o n decreases s i g n i f -i c a n t l y f o r both methylation processes. In the f i r s t methylation series of Part 3 no s i g n i f i c a n t decrease i n the extent of s u b s t i t u t i o n was ob-served for reaction times of from 3,75 to 30 minutes. The methoxyl contents of the four products were a l l within 0,2$, In the second methylation series a s i g n i f i c a n t decrease i n methoxyl content was found when the reaction time was reduced to 5 minutes or l e s s (Fig, 3), The r e s u l t s show that i n the f i r s t series methyl-a t i o n p e r s i s t e d during d i a l y s i s and, as a r e s u l t , no mini-mum time i n t e r v a l was observed. On the other hand, i n the second seri e s wherein the reaction was stopped before d i a l y s i s , a minimum time i n t e r v a l was observed. Taken to-gether these r e s u l t s show two things. One i s that the methylation reaction may be stopped or considerably reduced by n e u t r a l i z i n g the a l k a l i present. The second i s that, under the conditions used, at room temperature, no gain i n extent s u b s t i t u t i o n r e s u l t s a f t e r about the f i r s t f i v e minutes of reaction. Results on the e f f i c i e n c y of methylation with v a r i a t i o n i n t o t a l reagent concentration (Part 4), i l l u s t -rated graphically i n Pig. 4, show that by increasing the i n i t i a l t o t a l reagent concentration the extent of s u b s t i -tution i s Increased. On the other hand, the extent of sub-s t i t u t i o n i s not as great f o r the single operation process as i t i s f o r the continuous process (Part 2). A comparison of the e f f i c i e n c i e s f o r the two processes brings out the same r e l a t i o n . The e f f i c i e n c y of the continuous process i n which a t o t a l o f 2.8 times the th e o r e t i c a l amount of d i -methyl sulphate was added equally a f t e r three 15 minute i n -t e r v a l s i s 2.5$ higher than f o r the single stage process i n which the same t o t a l amount of reagent was added i n i t i a l l y . Since the difference i n e f f i c i e n c y i s not great, i t follows that almost the same extent of s u b s t i t u t i o n may be reached i n shorter time by employing a large excess of reagent. I t i s possible that dropwise addition of reagents i n a con-tinuous methylation process would show a greater difference i n e f f i c i e n c y over the one stage process. -26-The eurve i n Pig. 5 "plotting the extent of sub-s t i t u t i o n against sodium hydroxide concentration i s almost l i n e a r f o r the most part. This shows c l e a r l y that the extent of s u b s t i t u t i o n i s very nearly proportional to the a l k a l i concentration. I t may be seen from the p l o t of e f f i c i e n c y against sodium hydroxide concentration (Fig. 5) that above a concentration of about 5$ sodium hydroxide the e f f i c i e n c y of reaction increases almost l i n e a r l y with a l k a l i concentra-t i o n . Beyond a concentration of about 20$ sodium hydroxide the gum does not form a f l u i d s o l u t i o n and intimate mixing of the gum with reagents i s hindered. I t i s to be expected, therefore, that the optimum concentration of sodium hy-droxide i s close to 20$. The a n a l y t i c a l r e s u l t s on a sample methylated by dropwise addition of sodium hydroxide show that t h i s process i s more e f f i c i e n t than the one stage process. In f a c t the e f f i c i e n c y i s comparable to methylation with 15$ sodium hy-droxide, although i t i s about 5$ l e s s e f f i c i e n t than the one stage process with 20$ sodium hydroxide. Also i t may be seen (Table 7) that a much longer time of reaction i s required f o r complete reaction where dropwise addition of a l k a l i i s made. Results of the second separate experiment wherein a buffer s o l u t i o n was employed show that no substitution can be effected under these conditions. A l l the dimethyl s u l -phate was u t i l i z e d i n Bide reactions so that the e f f i c i e n c y of the reaction was 0$. I t may be i n f e r r e d from t h i s r e s u l t that a s l i g h t degree of a l k a l i n i t y i s required or that at l e a s t a c e r t a i n amount of a l k a l i must be present i n order f o r -27-methylation to proceed. The e f f e c t of substituting ammonium hydroxide f o r sodium hydroxide was to reduce the reaction e f f i c i e n c y to 0%, This r e s u l t was unexpected i n view of the fact that Lolkema(27) has suggested the use of ammonium hydroxide with d i a l k y l sulphates f o r the methylation of starch. Prom a study of the heat of reaction of dimethyl sulphate with ammonium hydroxide (Fig, 7), however, i t would seem that the reason that no subs t i t u t i o n i s obtained i s because the hydrolysis side reaction occurs too quickly. I t i s possible that at lower temperatures (less than 20°) hydrolysis i s slower and methylation can occur. Results on the e f f e c t of methyl alcohol and d i -ethyl ether on methylation e f f i c i e n c y (Table 7) show that methyl alcohol reduces the methylation e f f i c i e n c y consider-ably. This reduction may be attributed to the side reaction between dimethyl sulphate and the methyl alcohol, since i t was observed that considerable pressure from dimethyl ether resulted during reaction. This side reaction i s probably involved even i n aqueous solvents to a large extent, since considerable pressure, presumably due to dimethyl ether, r e s u l t s during methylation. With d i e t h y l ether-water (1:2.) as solvent, on the other hand, the e f f i c i e n c y of reaction was higher by about 10$, I t may be that the ether-water solvent f a c i l i t a t e s contact of the water insoluble dimethyl sulphate reducing the heterogeneous nature of the reaction mixture, thereby f a c i l i t a t i n g s u b s t itution. In t h i s l i n e of investigation -28-i t would be i n t e r e s t i n g to determine the reaction e f f i c i e n c y i n other solvent p a i r s and the optimum solvent r a t i o . The curves shown i n Pig, 6, bring out several i n t e r e s t i n g facts. For one thing i t may be seen that hy-d r o l y s i s of dimethyl sulphate by water alone at 24° i s very slow. On the other hand, hydrolysis under the same condi-tions at 95° i s very rapid and i s more rapid the higher the concentration of dimethyl sulphate(42), I t may be seen also that dimethyl sulphate i n 2$ sodium hydroxide solu t i o n over the range 24-34° loses only one methyl group by hydrolysis. With 11.1$ a l k a l i over the range 25-54° almost 10$ of the second methyl group i s l o s t i n 20 minutes. At 95° , even i n d i l u t e sodium hydroxide, about 70$ of the dimethyl sulphate i s hydrolysed i n 15 minutes. The lower two curves show the influence of s a l t s on the rate of hydrolysis. Prom an exam-ina t i o n of a l l these curves i t would seem that the methyl-ation e f f i c i e n c y w i l l be aided by low temperatures (0-20°) i n the i n i t i a l stages of reaction, since at higher tem-peratures the i n i t i a l reaction i s probably too rapid f o r mehtylatlon to occur. On the other hand, i f use i s to be made of the second methyl group i n dimethyl sulphate, higher temperatures are required. I t follows also that the higher the concentration of a l k a l i employed f o r reaction the lower the temperature required f o r removal of the second methyl group, although the two are not inversely proportional. The detrimental e f f e c t of the presence of excess s a l t i n the -reaction mixture suggests that removal of spent reagents from time to time i s advisable. This i s i n agreement with the -29-findings i n Part 2 wherein the stepwise methylation process, involving removal of reagents a f t e r each step, was shown to be more e f f i c i e n t than continuous methylation, a process i n which the spent reagents are allowed to accumulate. The curves i n Pig, 7 and Pig, 8 showing the v a r i a -t i o n i n temperature of reaction mixtures with reaction time bring out several f a c t s . In the f i r s t place i t may seen that there i s a good c o r r e l a t i o n between heats of reaction and rate of hydrolysis. With 2% sodium hydroxide the maximum temperature reached i s about 34°, while with 20$ a l k a l i the maximum i s about 62 , I t may be seen also that the more concentrated the a l k a l i s o l u t i o n the greater i s the l a g i n reaction. This suggests that with concentrated a l k a l i solu-tions a longer reaction time may be required or the reaction may require i n i t i a t i o n by heating. The fac t that the ammon-ium hydroxide curve reaches a maximum of 71° i n about one minute supports the conclusion reached i n Part 5, that side reaction occurs too quiekly f o r methylation to take place. Pig, 8 shows that the heat of reaction i s just as pronounced i n the presence of carbohydrate material as i n i t s absence. The scattered points i n Pig. 9 indicate that there i s l i t t l e c o r r e l a t i o n between the s p e c i f i c r o t a t i o n of par-t i a l l y mesquite gum products and the methoxyl content. I t would seem that there i s some decrease i n s p e c i f i c rotation with increasing methoxyl content but the r e l a t i o n i s not well defined. This i s not i n agreement with the findings of Haworth and Percival(26) who state that "the determination of -30. s p e c i f i c r o t a t i o n of methylated polysaccharides, as well as being more convenient, i s more sensitive as a control of methoxyl content than Z e l s e l estimations". The lack of c o r r e l a t i o n may re s u l t from two causes. One i s that measure-ments were made i n d i l u t e s o l u t i o n (2%) with a short tube (0.5 dm.) to allow s u f f i c i e n t l i g h t penetration and, as a r e s u l t , small errors i n measurement are greatly magnified (100 times). The second i s that p a r t i a l l y methylated pro-ducts were probably not homogeneous with respect to meth-oxyl content. I t i s possible that s p e c i f i c r o t a t i o n measure-ments on methylated gum samples i n other solvents, such as chloroform, may be more s a t i s f a c t o r y . However, i t may be that f r a c t i o n a t i o n of the gum i s necessary f o r t h i s type of measurement, SUMMARY AND CONCLUSIONS It was found that stepwise methylation of mesquite gumcsamples was more e f f i c i e n t than continuous methylation. In both processes, as the degree of sub s t i t u t i o n increases to about 30$ methoxyl content, the rate of s u b s t i t u t i o n de-creases considerably. The minimum time of methylation f o r equivalent s u b s t i t u t i o n was variable depending upon the reaction condi-tions but appears to be short, i n the v i c i n i t y of 5 minutes. I t was found that, with the same t o t a l quantities of reagents, the e f f i c i e n c y of reaction f o r continuous methylation i s a l i t t l e higher than that f o r a single stage methylation process. This implies that, i n continuous methylation, the time required to reach a given degree of s u b s t i t u t i o n i s roughly inversely proportional to the amount of reagents used. For a f i x e d r a t i o of dimethyl sulphate to mesquite gum (l.S3:l) and a f i x e d molar r a t i o of sodium hydroxide to dimethyl sulphate ( l . l : l ) , i t was found that the degree of s u b s t i t u t i o n i s p r a c t i c a l l y proportional to the a l k a l i con-centration with a probable optimum close to 20% sodium hy-droxide, Dropwise addition of 30$ sodium hydroxide to mes-quite gum solu t i o n i n the presence of dimethyl sulphate proved to be more e f f i c i e n t than the method of periodic addi-t i o n employed i n continuous methylation. From the r e s u l t s of methylation i n a buffer solut i o n i t i s concluded that a s l i g h t degree of a l k a l i n i t y or at l e a s t the presence of a l k a l i i s required f o r methyl-ation to take place. At room temperature ammonium hydroxide i s an un-s a t i s f a c t o r y substitute f o r sodium hydroxide, since side re-actions with dimethyl sulphate occur exclusively. For the above reason methyl alcohol-water proved to be an unsatisfactory solvent f o r the methylation reaction. I t i s suggested that the formation of methyl alcohol by side reaction during methylation i s a chief f a c t o r contributing to low methylation e f f i c i e n c i e s . The use of d i e t h y l ether-water (1:2) as a solvent f o r methylation was found to increase the reaction e f f i c i e n c y s i g n i f i c a n t l y . The increase i s attributed to a gain i n gum to solvent to dimethyl sulphate compatibility. At room temperature side reaction by aqueous hydrolysis of dimethyl sulphate i s not appreciable. At temperatures i n the range 20-50° a l k a l i hydrolysis i s re- , s t r i c t e d l a r g e l y to attack on only one methyl group i n d i -methyl sulphate. At 95° i n i t i a l aqueous and a l k a l i n e hy-d r o l y s i s i s very rapid and both methyl groups are hydrolysed. The deleterious e f f e c t of large quantities of s a l t (Na 2S0 4) on hydrolysis suggests that removal of spent reagents at suitable i n t e r v a l s during methylation i s advantageous. The saponification of dimethyl sulphate i s an exothermic reaction causing a considerable evolution of heat during methylation. Heat of reaction was found to increase with a l k a l i concentration and p a r a l l e l s the rate of hy-d r o l y s i s . I t i s suggested that too rapid a rate of hy-d r o l y s i s with a proportionate l i b e r a t i o n of heat lowers the methylation e f f i c i e n c y by allowing i n s u f f i c i e n t time f o r re-action with s t a r t i n g material. No d i s t i n c t c o r r e l a t i o n between s p e c i f i c ro-t a t i o n and methoxyl content of p a r t i a l l y methylated mesquite gum products was found. Lack of c o r r e l a t i o n i s attributed to the c o l l o i d a l nature of the methylated products i n water, making measurement of s p e c i f i c rotation uncertain, and to the inhomogeneity of samples with respect to methoxyl content. F i n a l l y , i t may be said that, from the re s u l t s of studies on the dimethyl sulphate-alkali methylation of mesquite gum, c e r t a i n guiding p r i n c i p l e s conducive to e f f i -cient methylation of water-soluble carbohydrates are apparent. -33-In the f i r s t place, a high concentration of a l k a l i i s de-s i r a b l e . I f reagents are added dropwise, then an amount of water just s u f f i c i e n t f o r so l u t i o n i s desirable. In the second place, i t i s advantageous to employ an excess of r e -agents amounting to 2 or 3 times the th e o r e t i c a l quantities, the In t h i r d place, removal of reagents and repeated methylation i s desirable when the methylation reaches an advanced degree of substitution. These p r i n c i p l e s set f o r t h are not new but serve to support those which were adopted i n the numerous methylation studies c a r r i e d out by Haworth(20). Further study of the methylation r e a c t i o n at various temperatures and i n the presence of various solvents may serve to define more clos e l y the optimum conditions f o r e f f i c i e n t dimethyl sulphate methylation. -34-BIBLIOGRAPHY lo B e l l , D.J. . • • . . Annual Rev. Biochem., 18,87(1949). 2. Haworth, W.N. and Machemer • . . . . J. Chem. Soc., 134, 2,270,2,372(1932), 3. Jeanloz, R Helv. Chim. Acta 27,1509(1944). 4. Spurlin, H.M . J . Am. Ghem. Soc, 61,2222(1939). 5. Bolliger, H.R., and Prins « . . . .Helv. Chim. Acta 28, 465(1945). 6. Timell, T Svensk Papperstidn. 51,52(1948). 7. Timell, T i b i d . 51,199(1948). 8. Timell, T ibid. 51,509(1948). 9. Timell, T. . . . . . Svensk Kem. Tid. 61,49(1949). 10. Haskins, J.F Advances i n Carbohydrate Chem., vol. 2, 1946. 11. De Bucear, M Papeterie 69,202(1947). 12. Sonnerskog, S Tek. Tid. 77,133(1940). 13. Radley, J.A Paint Manuf. 17,83(1947). 14. Purdie, T. and Irvine J. Chem. Soc, 1021(1903). 15. Irvine, J.C., Pringsheim and Skinner 1 I. I. 1 ~i. Ber.6SB, 2372(1929). 16. Smith, F. I • • • • J. Ghem. Soc, 510(1944). 17. Gakhokidze, A.M. . . . . • J. Appl. Chem. (U. S. S.R. )19, 1197(1946). 18. Schlmbach, H.H., and Huchting. . . . . Ann. 561(1949). Id. Denham, W.S. and Woodhouse . . . . .J. Ghem. Soc 105, 2357(1914). 20. Haworth, W.N J. Chem. Soc 107,8(1915). 21. Hendricks, B.C. and Rundle . . . . . J. Am. Chem. Soc 60, 2563(1938). 22. Pacsu, E. and Trister J. Am. Chem. Soc 61, 2442(1939). 23. Haworth, W.N. , H i r s t and Thomas . . . . . J , Ghem. Soc. 821(1931). 24. Maxwell, S.W. . . . . .U.S. Pat. 2,101,263(1937). 25. Haworth, W.N., H i r s t and Webb i l . . . J . Ghem. Soc. 2681(1928). 26. Haworth, W.N. and P e r c i v a l i b i d . 2277(1932). 27. Lolkema, J U.S. Pat. 2,459,108(1949). 28. Preudenberg, K. and Hixon Ber. 56,2119(1923). 29. Muskat, I.E. . . . . . J . Am. Ghem. Soc. 56,2449(1934). 30. Preudenberg, K. and Boppel Ber. 73,609(1940). 51. Hess, K., Schulze and Krajnc Ber. 73,1069 32. Meyer, K.H. and Gurtler . . . . . Helv. Ghim. Acta 31, 100(1948). 33. Schoriginia, N 34. White, E.V. i. 35. White, E.V. . 36. White, E.V. . 37. White, E.V. . N . . . . J . Gen. Ghem. (U.S.S.R.) 14, 825(1944). • J . Am. Chem. Soc. 68,272(1946). . i b i d . 69,622(1947). . i b i d . 69,2264(1947). . i b i d . 70,367,(1948). 38. Cunneen, J . I . and Smith J . Chem. Soc. 1141(1948). 39. Anderson, E. and Otis . . . . . J . Am. Chem. Soc. 52,4461, (1930). 40. Viebock, P. and Schwappach . . . . . Ber. 63B, 2818(1930). 41. Clark, E.P J . Assoc. O f f i c i a l Agr. Ghem. 15, 136(1932). 42. Lewis, H.P., Mason and Morgan Ind. Eng. Chem. 16,811(1924). APPENDIX Sample calculations of methylation e f f i c i e n c y : Example 1. The average e f f i c i e n c y of reaction f o r products 1 and 2 i n series 1 i s computed. Average methoxyl content a f t e r methylation = 15.45 Methoxyl content before = 2.48 Increase i n methoxyl content = 13.0 Molecular weight of s t a r t i n g material = 1061 Volume of dimethyl sulphate used =5.0 ml. I f x methoxyl groups are present i n the product, i t follows that x-1 methoxyl groups were introduced, since one group i s present i n the s t a r t i n g material. The expression f o r the methoxyl content of the product becomes: 31.03 x = 15.45 1061 + (x-1} 14.02 100 Solving, x = 5.6, x-1 =4.6 groups Thus the new molecular weight = 1125 and the t h e o r e t i c a l y i e l d = 5.3 g. or 106$ Assuming t h e o r e t i c a l y i e l d , since 4.6 methoxyl groups were introduced per mole of gum, i t follows that 4.6 x 31.03 x 5.0 grams were introduced. 1061 - i -Assuming that one mole of dimethyl sulphate i s re-quired for the introduction of one mole of methoxyl, then the amount of dimethyl sulphate u t i l i z e d i s given by 4.6 x 31.03 x 5.0 x 126.13= 2.75 g. = 2.05 ml. 1061 31.03 Thus the e f f i c i e n c y of the reaction expressed as a percentage i s 2.05 x 100 = 41.0$ 5.0 Example 2. The average e f f i c i e n c y of reaction f o r products 3 and 4 i n series 1. Prom Tables 1 and 2 the average increase i n methoxyl content i s 21.32$ The expression f o r the methoxyl content of the product i s given by 31.03 x = 25.8 1061 + (x-1) 14.02 100 from which x =9.0, x-1 = 8.0 groups This corresponds to 4.76 g. = 3.57 ml. Me 2S0 4 Since a t o t a l of 10 ml. of dimethyl sulphate were used, the o v e r a l l e f f i c i e n c y i s 3.57 x 100 = 35.7$ 10.0 I t was shown i n the previous sample c a l c u l a t i o n that the reagent e f f i c i e n c y during the f i r s t hour of methylation i s 41.0$. I t follows then that the e f f i c i e n c y during the second hour i s given by 41.0 + x = 35.7 2 from which x = 30.4$ Example 3. Cal c u l a t i o n of the maximum methoxyl content of completely methylated mesquite gum. I f the structure page 5 i s assumed, then i t may be seen that 17 hydroxyl groups, including the ga l a c t o s i d i c hy-droxyl group, are available for methylation. I f these are methylated then there are 18 methoxyl groups per mole present. The equation f o r the maximum methoxyl content becomes x = 31.03 x 18 100 1061 + (17 x 14.02) from which x - 43.0$ methoxyi Example 4. Ca l c u l a t i o n of dimethyl sulphate necessary f o r complete methylation of 5 g. mesquite gum. I f i t i s assumed that one mole of dimethyl sulphate i s required f o r one mole of hydroxyl, then we require 17' x 5 moles of dimethyl sulphate s 17 x 5 x 126.1 grams 1061 1061 s 17 x 5 x 126.1 = 7.58 ml. 1061 1.332 - i i i -

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