A COMPARATIVE STUDY OF THE HYDROLYSIS 
 OF CERTAIN ESTERS BY LIPASE 
 
 By 
 
 ELIZABETH CHARLOTTE HYDE 
 
 B. A. Mt. Holyoke College, 1909 
 
 THESIS 
 
 SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS 
 FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY 
 IN THE GRADUATE SCHOOL OF THE 
 UNIVERSITY OF ILLINOIS, 1922 
 
 URBANA, ILLINOIS 
 
Digitized by the Internet Archive 
 in 2016 
 
 https://archive.org/details/comparativestudyOOhyde 
 

 UNIVERSITY OF ILLINOIS 
 THE GRADUATE SCHOOL 
 
 .,ar 23 __ .192 ° 
 
 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY 
 
 SUPERVISION BY Izajaal ii. Chari ott e I£.dj3 
 
 ttxttttt .ff> A COMPARATIVE STUDY Off THE HYDROLYSIS OF CBR MIIL 
 
 ESTERS BY LIPASE 
 
 BE ACCEPTED AS FULFILLING THIS PART OF THE REQUIREMENTS FOR 
 THE DEGREE OF ...ASTER OF SCIEN CE 
 
 Recommendation concurred in* 
 
 Committee 
 
 on 
 
 Final Examination* 
 
 ^Required for doctor’s degree but not for master’s 
 
 499016 
 

 
 
 
 
 
 
 
 ■ I 
 
 
 
The writer is glad to have this 
 opportunity to acknowledge her indebted- 
 ness to Dr. Howard B • Lewis for his kind, 
 assistance in this research. 
 
TABLE OS’ CONTENTS 
 
 I GENERAL INTRODUCTION 1 
 
 II HISTORICAL ... ..... 2 
 
 III EXPERIMENTAL, PART I ..... 4 
 
 IV EXPERIMENTAL, PART II 15 
 
 V SUMMARY 32 
 
 VI BIBLIOGRAPHY 34 
 
I INTRODUCTION 
 
 Enzymic reactions are important in physiological processes. 
 It is possible to trace most processes of anabolism and catabol- 
 ism to enzyme action, but there our knowledge ceases because 
 the nature and mode of the action of enzymes is not thoroughly 
 understood. To explain physiological processes from a chemical 
 standpoint it is necessary to understand the kinetics of the 
 various enzyme reactions. It has been the ©eject of this study 
 to investigate under carefully controlled conditions the mode 
 of action of the fat-splitting enzymes or lipasis on certain 
 types of esters. The results reported here are concerned, with 
 the action of liver lipase on certain esters of the dicarboxy- 
 lic acid 3. 
 
 

 II HISTORICAL 
 
 Certain facts have been obtained regarding the phenomena j 
 of the hydrolytic action of lipase 021 esters of dicarboxylic j 
 acids. Kastle(l) in 1902 found that diethyl succinate was read-' 
 ily hydrolysed by liver lipase but he offered no quantitative 
 data as to the extent or products of hydrolysis. He also ob- 
 served that the metallic salts of the monoethyl esters such as 
 potassium ethyl succinate and sodium ethyl succinate were not 
 split by lipase. The inactivity of lipase toward this class of 
 compounds was explained by Kastle as being due go the ionization 
 of the metallic salts. He believes that ionized compounds are 
 stable toward lipase. 
 
 In 1903 Doyen and Morel(2 ) observed that serum lipase could 
 hydrolyse diethyl succinate. However they did not isolate the 
 products of hydrolysis. 
 
 Morel and Terronie( 5 ) undertook a comparative study of the 
 hydrolysis of the diethyl esters of malonic, succinic, glutanic, 
 suberic and sebacic acids by the pancreatic juice of the dog 
 and concluded that the ease of hydrolysis increased with the 
 increase of the molecular weight of the acid up to glutaric 
 acid. The maximum hydrolysis obtained by them for diethyl mal- 
 onate and diethyl succinate were 2.6 per cent and 12.9 per cent 
 respect ively • 
 
 Christman and Lewis(6) made a careful study of the action 
 
 of liver lipase on various esters, including ethyl succinate and 
 

 
 
 - 
 
 
 
 
 / 
 
 
 
 
 
 
 
 
 
 
 I * 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
- 3 - 
 
 ethyl malonate. Kastle's observation regarding the stability 
 of the metallic salts of the monoethyl esters of the dicarboxy- 
 lic acids was confirmed by their experimentation. Their results 
 also seem to snow that the hydrolysis of the diethyl esters of 
 malonic and succinic acids can proceed only to the removal of 
 one ethyl group and that the monoethyl esters as well as their ! 
 metallic salts are relatively stable toward lipase. 
 
- 4 ™ 
 
 ITT EXPERIMENTAL 
 
 Lipase may act in three ways in the hydrolysis of an ester 
 of a dicar'box 3 r lic acid. One ethyl group my be removed and 
 the second ethyl group left intact. One ethyl group may be 
 removed first and the second ethyl group subsequently attacked, 
 or both ethyl groups may be removed simultaneously by the 
 lipase. It seems quite probable that lipase attacks different 
 esters in a different manner. 
 
 To determine the mode of lipolytic hydrolysis it is nec- 
 essary to find out what products result from the reaction. 
 
 This research has been divided into two main problems, the 
 first being a study of the time required for the diethyl esters 
 of dicarboxylic acids to attain an equilibrium in their hydro- 
 lytic reaction with liver lipase under varying conditions of 
 acidity, and the second an attempt at the isolation of the 
 products of these reactions. 
 
 The esters studied in this manner were diethyl malonate, 
 diethyl succinate, diethyl glutarate and diethyl adipate. A 
 study of the hydrolysis of ethyl propinnate by liver lipase 
 was made in order co determine to what extent the hydrolysis 
 of a simple ester could be effected under the conditions of 
 these experiments. 
 
 The lipase extract which was used in this research was 
 prepared from a liver taken from a hog immediately after the 
 death of the animal. According to the method of Kanitz( 3 ) 
 
- 5 - 
 
 two parts of glycerol were added to one part of the finely cnop- 
 ped tissue. After a thorough shaking the mixture was allowed 
 to stand at least a week and then filtered through cheesecloth 
 when needed. The extract retained its constant lipolytic action 
 unimpaired for several months. 
 
 Trie diethyl propionate, diethyl malonate, and diethyl succin- 
 ate used were commercial products. The diethyl glutarate was 
 made in the organic laboratory while the diethyl adipate was 
 prepared from adipic acid made by the method of Bouvealt and 
 Locquin(4) in the laboratory in which this research was carried 
 on. The purity of the esters used was determined by means of 
 saponification values. To obtain optimum conditions for the 
 action of lipase it is necessary that the concentration of the 
 ester be such that complete solution is effected, therefore 
 
 ■JT 
 
 ^T, and concentrations were used. 
 
 5cc portions of the glycerol extract were measured by means 
 of a pipette into 200 cc Erlenmeyer flasks. To each flask was 
 added 25 cc of the standard ester solution. These flasks were . 
 incubated at room temperature for varying periods of time. The 
 acidity developed by the reaction was then titrated with ^ 
 NaOH, using phenolthaiein as an indicator. The flasks were ar- 
 ranged in pairs and one pair was titrated after 15 minutes and 
 a second pair after minutes, etc. Each pair of flasks was 
 also retitrated at the intervals shown in the tables. The fig- 
 ures given in the tables represent the total volume of Ha OH 
 
 required for neutralization and are the averages of check deter- 
 
- 6 - 
 
 minations from which the blanks due to the acidity of the ester 
 solution and lipase extract have been subtracted. The total 
 acidity of the blank was at no time equivalent to more than .3 
 cc of yy II a OH. The left hand figures in each line of the 
 tables represents the average acidity developed in two flasks 
 which were first titrated at the end of the time stated at the 
 head of the column in which the figures are found. All figures 
 to the right represent the total acidity which had developed 
 during the interval of time stated at the top of the column in 
 which the figures are found. 
 

TABLE I 
 
 Hydrolysis of 0.025 F ethyl propionate by lipase. 25 cc por- 
 tions of ester were used. Bor the complete saponification of 
 this amount of ester 5*3 cc of 0,09872 F FaOH were required. 
 
 Time 
 
 30 ’ 1 hr 
 
 2 hr e 
 
 3 hrs 
 
 4 hrs 
 
 9 hrs 
 
 6 hrs 
 
 10 hrs 
 
 11 hrs 
 
 Standard FaOH 
 
 required for 
 
 neutralizat ion 
 
 of acidity 
 
 
 3-05 4.7 
 
 5.05 
 
 5 .O 7 
 
 5 .O 7 
 
 5.07 
 
 5.07 
 
 5.07 
 
 5.07 
 
 3,72 
 
 4.7 
 
 4.8 
 
 4,8 
 
 4.8 
 
 4,8 
 
 4.8 
 
 4.8 
 
 
 4.27 
 
 4,45 
 
 4.55 
 
 4.59 
 
 4.55 
 
 4.55 
 
 4.55 
 
 
 
 4.35 
 
 4.55 
 
 4.95 
 
 4.55 
 
 4.55 
 
 4.55 
 
 
 
 
 4.92 
 
 9.17 
 
 5.17 
 
 5.17 
 
 5.17 
 
 4.97 
 
 5.02 
 
 5.09 
 
 5.02 
 
 5.05 
 
 5.00 
 
 5.02 
 5.05 
 5,00 
 5 .15 
 
—3- 
 
 TABLE II 
 
 Hydrolysis of 0,05 IT diethyl malonate by lipase. 25 cc portions) 
 of ester were used. For trie complete saponif ication of this 
 
 amount 
 
 of e 
 
 ster 12 
 
 .6 c c 
 
 0 
 
 •b 
 
 0 
 
 • 
 
 09872 
 
 IT Ha OH 
 
 were 
 
 required . 
 
 
 Time 
 
 30’ 
 
 1 hr 
 
 2 hrs 
 
 3 hrs 
 
 4 hrs 
 
 5 hrs 
 
 6 hrs 
 
 7hrs 
 
 8 hrs 
 
 9 hrs 
 
 Cone . 
 of 
 
 Ester 
 
 2.0 
 
 3.62 
 
 5*52 
 
 6.0 
 
 6.25 
 
 6. 3 
 
 6.4 
 
 6.4 
 
 6.4 
 
 6.4 
 
 
 
 3*3 
 
 5*57 
 
 6.07 
 
 6.22 
 
 6.25 
 
 6.25 
 
 6.25 
 
 6.25 
 
 6*3 
 
 
 
 
 4.75 
 
 5*75 
 
 6.1 
 
 6.25 
 
 6.25 
 
 6.25 
 
 6.25 
 
 6,3 
 
 
 
 
 
 5*20 
 
 5.82 
 
 6.12 
 
 6.12 
 
 6.12 
 
 6.12 
 
 6.12 
 
 .05 H 
 
 
 
 
 
 5*55 
 
 6.02 
 
 6.22 
 
 6.55 
 
 6.55 
 
 6.4 
 
 
 
 
 
 
 
 5.6? 
 
 6.1? 
 
 6.45 
 
 6.47 
 
 6.52 
 
 
 
 
 
 
 
 
 5*3 
 
 6.0 
 
 6.3 
 
 6.35 
 
 
 
 
 
 
 
 
 
 
 5*9 
 
 6.1 
 
 
 
 
 
 
 
 
 
 
 6.1 
 
 6.1 
 
 

 
 
 
 
Hydro 
 29 ec 
 tion 
 were 
 
 -9- 
 
 TA3LE III 
 
 lysis of 0,05 and 0.023 11 diethyl 
 portions of ester were used. For 
 of this amount of ester 12.2 and 6 
 respectively required. 
 
 succinate hy lipase, 
 the complete saponifica- 
 .00 cc of 0.09872 N Ha OH 
 
 L 
 
 Time 
 
 
 
 
 
 
 Cone of 
 
 30’ 
 
 1 hr 2 hrs 
 
 3 hrs 
 
 4 hrs 
 
 9 hrs 
 
 7 hrs 
 
 9 hrs Ester j 
 
 
 Standard Ha OH 
 
 required for 
 
 neutralization 
 
 of acidity 
 
 : 
 
 2.67 
 
 5-15 5.70 
 
 9.72 
 
 9.72 
 
 S.72 
 
 3 . 73 
 
 5-75 
 
 
 4.32 5.97 
 
 6.02 
 
 6.02 
 
 6.02 
 
 6.07 
 
 6,07 
 
 
 5.7 
 
 6.02 
 
 6.02 
 
 6.02 
 
 6.03 
 
 6.09 
 
 
 
 5.9 
 
 6 . 09 
 
 6.03 
 
 6 . 03 
 
 6,03 0.09 u 
 
 
 
 
 6.07 
 
 6.1 
 
 6.1 
 
 6.1 
 
 
 
 
 
 6. '03 
 
 6.19 
 
 6.19 
 
 
 
 
 
 
 6.13 
 
 6.13 
 
 
 
 
 
 
 
 6.2 
 
 Time 
 
 
 
 
 
 
 Cone, of 
 
 30» 
 
 1 hr 2 hrs 
 
 3 hrs 
 
 4 hrs 
 
 6 hrs 
 
 8 hrs 
 
 9 hrs Ester 
 
 
 Standard Ha OH 
 
 requir 
 
 ed for 
 
 neutralization 
 
 of acidity 
 
 1*95 
 
 “2T73 ~1T 
 
 2 
 
 2,8 
 
 2.8 
 
 2,8 
 
 273 — 1 
 
 
 2.61 2.91 
 
 2.91 
 
 2.91 
 
 2.91 
 
 2,91 
 
 2.91 
 
 
 2.82 
 
 2.8? 
 
 2.87 
 
 2.87 
 
 2. 8? 
 
 2.87 
 
 
 
 2.9? 
 
 2.97 
 
 2,97 
 
 2,97 
 
 2.97 C.023 IT 
 
 
 
 
 2.97 
 
 2.97 
 
 2.97 
 
 2.97 
 
 1 
 
 
 
 
 
 2.9 
 
 2,9 
 
 2.9 
 
 
 
 
 
 
 3.07 
 
 3.07 
 
 
 
 
 
 
 
 3*09 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
VM 
 

 
 
 
 
 
 
 
 
 
- 11 - 
 
 TABLE V 
 
 Hydrolysis of 0,025 H solutions of diethyl adipate by lipase. 
 
 25 cc portions of the ester soluttion were used, 
 saponification of this amount of ester 6.35 cc 0 
 HaOH were required. 
 
 Bor complete 
 f 0.09872 IT 
 
 I 
 
 Time 
 
 30' 
 
 1 hr 
 
 2 
 
 hrs 
 
 3 hrs 
 
 4 hrs 
 
 5 hrs 
 
 7 hrs 
 
 9 hr 
 
 s 11 hrs 
 
 
 Standard 
 
 NaOH 
 
 required 
 
 for 
 
 neutral 
 
 izat i on 
 
 of aci 
 
 dity 
 
 2.7 
 
 3*97 
 
 4, 
 
 12 
 
 4.12 
 
 4.12 
 
 4.12 
 
 4,12 
 
 4.12 
 
 4,12 
 
 
 3.95 
 
 4. 
 
 45 
 
 4.45 
 
 4.45 
 
 4.45 
 
 4,45 
 
 4.45 
 
 4.45 
 
 
 
 5. 
 
 «S5 
 
 3.55 
 
 5*55 
 
 5*55 
 
 5*55 
 
 5*55 
 
 5*55 
 
 
 
 
 
 5 . 7 O 
 
 5.70 
 
 5*70 
 
 5.70 
 
 5.70 
 
 5*70 
 
 
 
 
 
 
 6.10 
 
 6.10 
 
 6.10 
 
 6.10 
 
 6.10 
 
 
 
 
 
 
 
 6.10 
 
 6.10 
 
 6.10 
 
 6.10 
 
 
 
 
 
 
 
 
 6.30 
 
 6,30 
 
 6.30 
 
 
 
 
 
 
 
 
 
 6.15 
 
 6.15 
 
 6.15 
 
 Time 
 
 
 
 
 
 
 
 
 
 
 15’ 
 
 30 . 
 
 45 
 
 • 
 
 1 hr 
 
 2 hrs 
 
 3 hr s 
 
 4 hrs 
 
 8 hrs 
 
 10 hrs 
 
 
 Standard 
 
 ITaOH 
 
 recxuired 
 
 for 
 
 neutralizati on 
 
 of acidity 
 
 1.32 
 
 2,70 
 
 3* 
 
 7 
 
 3.82 
 
 3.82 
 
 3.86 
 
 3*85 
 
 3*85 
 
 3*85 
 
 
 1.90 
 
 3. 
 
 0 
 
 3.15 
 
 3*2 
 
 3.25 
 
 3*3 
 
 3*3 
 
 3*32 
 
 
 
 3- 
 
 82 
 
 4 , 32 
 
 4.40 
 
 4.40 
 
 4.5 
 
 4.5 
 
 4.5 
 
 
 
 
 
 3.87 
 
 4.62 
 
 4.65 
 
 4.65 
 
 4.65 
 
 4.65 
 
 
 
 
 
 
 5*0 
 
 5 ..12 
 
 5*12 
 
 5.12 
 
 5 . 12 
 
 
 
 
 
 
 
 5*85 
 
 5.85 
 
 5.83 
 
 5.85 
 
 
 
 
 
 
 
 
 5*9 
 
 5*9 
 
 5*9 
 
 
 
 
 
 
 
 
 
 6,03 
 
 6.03 
 

 
 
 
 
 
- 12 - 
 
 TABLE Y-c ont inu ed 
 
 Hydrolysis of 0.0125 i\ r solution of diethyl adipate by lipase. 
 
 25 cc portions of the ester solution were used. Ecr complete 
 saponification of this amount of ester 3*2 cc of 0,09872 ]J KaOH 
 
 were required , 
 
 Time 
 
 30 * 
 
 1 hr 2 
 
 hrs 
 
 3 hrs 4 
 
 hrs 
 
 5 hrs 6 hrs 8 hrs 9 hrs 
 
 
 Standard 
 
 NaOH 
 
 required 
 
 f or 
 
 neutralization of acidity 
 
 1.425 
 
 2.10 
 
 2.10 
 
 2.15 
 
 2.15 
 
 2.15 
 
 2.15 
 
 2.15 
 
 *6? 
 
 2 7 
 
 2.32 
 
 2 .32 
 
 2,32 
 
 2.32 
 
 2,32 
 
 2.32 
 
 
 1.05 
 
 2.45 
 
 2.45 
 
 2.45 
 
 2.45 
 
 2.45 
 
 2.4S 
 
 
 
 1.85 
 
 2.25 
 
 2.25 
 
 2.25 
 
 2.25 
 
 2.25 
 
 
 
 
 2.1 
 
 2.55 
 
 2.55 
 
 2.55 
 
 2.55 
 
 
 
 
 
 2.25 
 
 2.35 
 
 2*35 
 
 2.35 
 
 3*075 3*075 
 
 3*35 
 
-13- 
 
 Ethyl propionate .-Data presented in Table I show that 
 ethyl propionate can be h3 r drolysed by lipase to the extent of 
 90 per cent under the conditions of this experiment. 
 
 Diethyl malonate .-The results of the experiment with di- 
 ethyl malonate as substrate, Table II, are in harmony with 
 those of Christman and Lewis( 6 ) in that they show that the hy- 
 drolysis by lipase proceeds only half way to completion under 
 the conditions of this experiment. A later experiment of a 
 different type indicates that the extent of hydrolysis can be 
 increased about 5 ° per cent by an increase in the dilution of 
 the reaction mixture, whether the dilution herein represented 
 by the addition of 6 cc of NaOH or a 25 per cent increase 
 is comparable to the 200 per cent increase in the later exper- 
 iment would have to be determined by further study. 
 
 Diethyl succinate .-The results of the hydrolysis of di- 
 ethyl succinate b3 r liver lipase, Table III, also correspond to 
 those obtained by Christman and Lewis(6). An equilibrium was 
 reached at the end of one or two hours when the acid produced 
 by the reaction had been neutralized at varying intervals. The 
 
 rate of hydrolysis increased up to the point of equilibrium 
 
 i, 1 ; 
 
 and after that time no increase was noted. In both “7 and 43 
 
 concentrations of diethyl succinate the hydrolysis was no 
 greater than 50 per cent of the theoretical. 
 
 1 
 
 Diethyl glutarate.-The 0.025 N solution of diethyl glutar- 
 a.te, Table IV, was completely hydrolysed by lipase, while the 
 hydrolysis of the 0.05 N solution, Table IV, went only about 
 three fourths of the way to completion. It would also appear 
 
- 14 - 
 
 from these data that when the acid produced by the reaction was 
 neutralized an equilibrium was reached when the hydrolysis had 
 proceeded about halfway to completion and beyond this point 
 hydrolysis was very slight* 
 
 Diethyl adipate. -The observation which stands out most 
 prominently in regard to the hydrolysis of diethyl adipate, 
 Table V, is that lipase can completely hydrolyse this ester. 
 From these data it may also be observed that when the acidity 
 produced by the reaction was neutralized an equilibrium was 
 reached after the hydrolysis had proceeded two thirds of the 
 way to completion and beyond this point there was apparently no 
 further hydrolysis. 
 
- 15 - 
 
 IV EXPERIMENTAL, PART II 
 
 Isolation of Products of Hydrolysis. 
 
 The second type of experimentation was an attempt to iso- 
 late the products of the hydrolysis of the esters "by lipase . 
 
 In order to isolate amounts of acid and ester sufficient for 
 identification it was necessary to ca rvy out the hydrolysis of 
 much larger amounts of the esters. 4 cc portions of pure ester 
 were measured into 700 cc Eflenmeyer flasks. 100 cc of HsO 
 and 5 cc of liver lipase were added. Two flasks were set up 
 for each ester studied. Toluene was added as a preservative 
 and care was taken to shake each flask frequently in order to 
 maintain an emulsion of the toluene. These flasks were incubat- 
 ed at room temperature for a period of two weeks or more. Dur- 
 ing this time the acidity' developed in one flask was neutraliz- 
 ed daily while the acid in the second flask was not neutralized 
 until the end of the period of incubation. 
 
 In order to determine the extent of hydrolysis of an ester 
 of a dicarboxylic acid it is necessary to separate the diethyl 
 ester if any remains from the monoethyl ester and the salts. 
 
 At the beginning of this investigation isolation of the products 
 of hydrolysis was effected by means of the method of Christman 
 and Lewis(6). The filtrate from the digestion mixture was care- 
 fully extracted several times with ether. This should have re- 
 moved any unchanged ester which would have been present in con- 
 siderable quantities if the hydrolysis of the esters studied 
 

16 - 
 
 had attacked both ethyl groups simultaneously. The ether ex- 
 tract was evaporated at room temperature and after solution of 
 the residue in water aliquots we re titrated to determine acid- 
 ity and saponified to determine the amount of saponifiable mat- 
 erial. The solution which remained after the ether extraction 
 was acidified with H2SO4 and repeatedly extracted with ether. 
 
 The combined ether extracts were washed several times and the 
 ether was removed by evaporation at room temperature. The re- 
 sidue was dissolved in HaO and aliquots were titrated and sapon- 
 ified. This portion should have contained the amid and any 
 monoethyl ester produced by hydrolysis. If the hydrolysis had 
 simply removed one ethyl group the amount of yw Ha OH necessary 
 to neutralize the acid and to saponify the monoethyl ester 
 should have been equal'. Table VI gives the results of digest ior 
 experiments in which this method of isolation of the products 
 was followed. 
 

 
 -17- 
 TABLE VI 
 
 
 
 
 The isolation of the products of hydroly 
 
 sis of diethyl 
 
 succ inat e 
 
 and diethyl 
 
 glutaraoe 
 
 "by the method of Christman 
 
 and Lewis. 
 
 Duration of 
 
 hydrolysis 
 
 17 days. 0.09872 
 
 N NaOH 
 
 ased for neutral- 
 
 ization and 
 
 saponif ica 
 
 t ion. 
 
 
 
 
 Standard 
 NaOH re- 
 quired for 
 complete 
 sap on if ic ac- 
 tion 
 
 Standard 
 ITaOH ac- 
 tually re 
 quired 
 
 Ether extract of filtrate 
 
 
 Before scidi- 
 - fication 
 
 After acidi- 
 f ication 
 
 Remarks 
 
 Standard ITaOH 
 to 
 
 Standard 
 
 to 
 
 NaOH 
 
 
 
 Neutral- Sap- 
 ize 
 
 Neutral- 
 
 ise 
 
 Sap- 
 
 onify 
 
 
 231 
 
 105.4 
 
 None 1.88 
 
 20.0 
 
 23 .I 
 
 2 c c of 
 diethyl 
 succinate 
 neutral is 
 ed at end 
 
 231 
 
 126.2 
 
 None 1,12 
 
 28.0 
 
 28.8 
 
 2 cc of 
 diethyl 
 succinate 
 neutraliz- 
 ed dally 
 
 463 
 
 144,4 
 
 None None 
 
 18.8 
 
 24.32 
 
 4 cc of 
 diethyl 
 succinate, 
 neutraliz- 
 ed at end 
 
 463 
 
 231.75 
 
 None None 
 
 68.8 
 
 69.44 
 
 4 cc of 
 diethyl 
 succinate 
 neut raliz- 
 ed dally 
 
 430 
 
 149.2 
 
 None 14.4 
 
 32.48 
 
 44.2 
 
 4 c c of d i 
 diethyl 
 glutarate 
 neutraliz- 
 ed at 1 
 
 430 
 
 309*1 
 
 None 25.2 
 
 41,20 
 
 40,0 
 
 4 cc of - " 
 diethyl 
 6 lu tar ate 
 neut raliz- 
 
 430 
 
 257*8 
 
 None 1.2 
 
 45*6 
 
 47.2 
 
 4 cc of 
 diethyl 
 glut a rat e 
 neutraliz- 
 ed daily 
 

-18- 
 
 Diethyl succinate, -The data on Table VI are of value in 
 that they show the extent hydrolysis of the esters under dif- 
 ferent conditions of acidity* The data regarding the products 
 of the hydrolysis point to inaccuracies in the recovery of acid 
 and ester. If lipase can remove only one ethyl group from di- 
 ethyl succinate then the reaction represented which was neutral- 
 ized daily had gone to completion. If both ethyl groups can be 
 removed by the lipase the same reaction has gone only half way 
 to completion. This fact cannot be determined from these data. 
 Th@ retardation of the hydrolysis reaction when the acid produc- 
 ed was not neutralized may have been due to the concentration 
 of the solution. Later experiments of the same type seem to 
 show that hydrolysis proceeds farther when K2O is added daily, 
 
 Diethyl glutarate.-It may be observed that under the condi- 
 tions of this type of experiment the hydrolysis of diethyl glu- 
 tarate proceeded only about half way to completion even when the 
 acid produced was neutralized frequently. The figures represent- 
 ing the daily titrations show that the hydrolysis proceeded very 
 rapidly at first and that an equilibrium was approximately reach- 
 ed at the end of the third day. It appears that when the acid 
 produced by the react! 021 was neutralised the hydrolysis readily 
 proceeded half way to completion while after that point was 
 reached the hydrolysis proceeded relatively slowly. 
 
 As the investigation progressed certain errors were suspect- 
 ed and therefore modifications were made in the method of re- 
 covery. TablesVII and VIII give the results of the hydrolysis 
 

 
 
 
 - 
 
 - 
 
 
 , 
 
 
 
 
 . 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
- 19 - 
 
 of diethyl succinate and the products isolated n oy modifications 
 of the method of Christiaan and Lewis(6)» 
 
“ 20 - 
 
 TABLE VII 
 
 Isolation of the products of hydrolysis of 4 cc of ethyl suc- 
 cinite by a slight modification of the method of Christman and 
 Lewis which consisted in salting cut with (NIU )aS04 before ex- 
 traction with ether. Duration of hydrolysis 40 days. 0.09872 
 N NaOH used for neutralization and saponification. 
 
 Standard 
 NaOH re- 
 quired for 
 
 Standard 
 NaOH ac- 
 tually 
 
 Ether ex 
 
 tract c 
 
 >f Filtrate 
 
 
 Before acid- 
 ification 
 
 After acid- 
 ification 
 
 Remarks 
 
 complete 
 saponif i- 
 c at ion 
 
 required 
 
 Standard 
 
 to 
 
 NaOH 
 
 Standard 
 
 to 
 
 NaOH 
 
 
 
 Neutral- 
 
 ise 
 
 Sap- 
 
 onify 
 
 Neutral- 
 
 ize 
 
 Sap- 
 
 onify 
 
 
 463 
 
 231 
 
 None 
 
 1.08 
 
 76.8 
 
 ?8,8 
 
 Neutral- 
 ised daily 
 
 463 
 
 165 
 
 None 
 
 14.4 
 
 61.6 
 
 6l,6 
 
 Neutral- 
 ized at end 
 
-21- 
 
 TABLE VIII 
 
 Isolation of the products of hydrolysis of 4 cc of diethyl 
 adipate by a modification of the method of Christman and Lewis 
 (6). This modification consisted in evaporating the filtrate 
 to dryness before extraction which was done with warmed ether. 
 Duration of hydrolysis IS days. 0.098/2 N La OH was used for 
 neutralisation and saponification. 
 
 Standard 
 HaOH re- 
 quired for 
 complete 
 saponifi- 
 cation 
 
 Standard 
 La OH ac- 
 tually 
 required 
 
 Ether Extract of filtrate 
 
 Remarks 
 
 Before acid- 
 . ification 
 
 After acid- 
 ification 
 
 Standard NaOE 
 to 
 
 
 neutral- 
 
 ise 
 
 Sap- 
 
 onify 
 
 400.1 
 
 401.2 
 
 Hone 
 
 Hone 
 
 Contained a 
 quantity of 
 white cr 3 T - 
 stals LLP. 
 14?. 6° H.E. 
 ?6 and no 
 saponiif ia- 
 ble material 
 
 neutralized 
 
 daily 
 
 400.1 
 
 152.5 
 
 Hone 
 
 Hone 
 
 Contained a 
 quantity of 
 white cry- 
 stals. M.P, 
 146° and sap- 
 onifiable mat- 
 erial equiva- v 
 lent to 6 
 HaOH 
 
 Neutralised 
 at end 
 
 400.1 
 
 209 
 
 4.0 
 
 Hone 
 
 Contained a 
 quantity of 
 white cry- 
 stals. M. P. 
 147 and sapon- 
 ifiable mater- 
 ial equivalent 
 to 34.4 cc — £ 
 HaOH 
 
 Neutralised 
 daily and re- 
 action stop- 
 ped when hydro 
 l 3 r sis he.d gone 
 half way 1 0 
 complexion 
 
- 22 - 
 
 Results presented in Table VIII are of interest in as much 
 as they show that diethyl adipate may be completely hydrolysed 
 b 3 T liver lipase when the acidity produced by the reaction is 
 neutralized daily*. The fact that the hydrolysis was greatly re- 
 tarded when the acid produced by the reaction was not neutral- 
 ized may have been due to the concentration of trie solution. It 
 is interesting to note that when the hydrolysis of 4 cc of di- 
 ethyl adipate had proceeded half way to completion as indicated 
 by the amount of standard HaOH which had been used to neutralize 
 the acid produced cryst als of adipic acid were obtained which 
 shows that the second ethyl group was attacked before the first 
 had been entirely removed* 
 
 Since the, theoretical yield of acid or ester had not been 
 obtained by ary modification of the method of Lewis and 
 Christman( 6 ) it was decided to try tc recover acid and ester 
 from known amounts of each under the same conditions as existed 
 J ^ ^ - digestions . Table IX contains the results of these 
 
 trials. 17 o quantitative recovery of either acid or ester was 
 
 i 
 
 obtained , 
 
 It was thought that the solid portions of the enzyme ex- 
 tract may ha., o adsorbed some of the ester and therefore if fil- 
 tration could be omitted one source of error would be eliminat- 
 ed. It also seemed advisable to eliminate the second ether ex- 
 -ractioii. To make this possible the solution from the first 
 ef.aer extraction was acidified with an excess of HC1 and evapor- 
 ated lc dryness on the steam bath. The residue was dissolved in 
 

- 23 - 
 
 HaO and aliquots wer titrated and saponified with stand trd 
 itfaOH to determine the amount of dicarboxylic acid and monoeth 
 ester present. 
 
 The next thought that suggested itself was the use of 
 CHCls as a solvent in place of ether. Tables X, XI, and XIT 
 give the results of some preliminary experiments carried out 
 in an effort to find a suitable method of isolation. 
 
 <.A 
 

-24- 
 TABLE IX 
 
 The isolation of succinic acid and succinic ester from a neu- 
 tralized solution containing 2.4 grams of the acid and 1.6 grams 
 of the ester by means of salting out with (HIU)2S04 and extract- 
 ing with ether before and after acidification. 
 
 2.4 grains of succinic acid are equivalent to 410. 3 cc O. 09 S 72 H 
 Ha OH 
 
 1.6 grams of succinic ester are equivalent to 182 cc 0,09872 H 
 
 IT a OH 
 
 Number 
 
 of 
 
 Exper- 
 
 iment 
 
 Ether Extract 
 
 of Solution 
 
 
 B ef or e ac id if i c at i on 
 
 After acidi: 
 
 f ication 
 
 Standard Ha 0E to 
 
 Standard Ha OH to 
 
 
 __ Neutralize 
 
 Saponify 
 
 Neutralize I 
 
 
 y 
 
 at. 
 
 Hone 
 
 48 cc 
 
 2,4 
 
 Hone 
 
 II 
 
 Hone 
 
 29.2 
 
 15.0 
 
 18 
 

 
 
 
 
 
 
 
 
 
 V. - 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
_0 £ „ 
 
 CL.sJ 
 
 Solutions of 
 with CKCls . 
 of 0.098^2 IT 
 
 TAB IT 3 ; XT 
 
 diethyl succinate and succinic acid were extracted 
 1 gram of succinic acid is equivalent to 171 cc 
 ITaOH. 1 gram of diethyl succinate is equivalent 
 
 to 114 cc of O.O 8872 N NaOH. 
 
 Number 
 
 of 
 
 Exp er- 
 
 iment 
 
 TT 30 Extraction 
 
 CKCls Extraction 
 
 Remarks 
 
 Standard ITaOH to 
 
 Standard NaOH to 
 
 — 
 
 Neutral- 
 
 ise 
 
 Sapon- 
 
 ify 
 
 Neutral- 
 
 ize 
 
 Sap on- 
 5 f 7 
 
 T 
 
 280.0 
 
 188.0 
 
 None 
 
 16.0 
 
 1 cc diethyl 
 succinate 
 1.5 grams suc- 
 cinic acid 
 
 II 
 
 185 . 0 
 
 102.0 
 
 Lost 
 
 Lost 
 
 1 cc ethyl suc- 
 c inat e 
 
 1 gr . succinic 
 acid 
 
 III 
 
 236 .O 
 
 214.0 
 
 2 cc 
 
 51.0 
 
 2 cc diethyl 
 succ inate 
 1.5 grams suc- 
 cinic acid 
 
-28- 
 
 As is apparent from a reading of the data given these 
 methods of isolation had not proven satisfactory. The greater 
 acidity obtained when the filtrate was acidified with HC1 and 
 evaporated to dryness may have been due to some residual HC1. 
 
 The larger amounts of saponifiable material obtained in the H3O 
 soluble portion when chloroform was used as a solvent may have 
 been due to decomposition of some dissolved chloroform in alka- 
 line reaction. It was also thought that during evaporation of 
 the chloroform on the steam bath steam distillation of the 
 ester may have taken place. 
 
 It seemed worth while to try vacuum distillation as a means 
 
 of separating the partially hydrolysed from the unhydrolysed 
 
 ester* Preliminary experiments using solutions of ester and acid 
 
 were carried out which proved that any diethyl ester present in 
 solution 
 
 a neutral /was carried over almost quantitatively in the distil- 
 late and that the monoethyl ester and the salts were left in 
 the residue apparently unchanged. In an attempt to obtain the 
 acid and monoethyl ester, the residue was acidified and extract- 
 ed with ether. Tables XTTI and XIV give the data obtained from 
 the preliminary experiments and Table XV the data ol • ined from 
 the use of this method in the isolation of the products of a 
 digestion b3 r lipase. 
 

- 30 - 
 
 TABLE XIV 
 
 One gram of diethyl succinate and one gram of succinic acid and 
 5 cc of boiled enzyme were neutralized aud distilled in vacuo. 
 The residue was acidified and extracted with ether. 
 
 1 gran of diethyl succinate is equivalent to 114 cc 0 . 09872 IT 
 
 Ha OH 
 
 1 gram of succinic acid is equivalent to 171 cc O.O 9872 H Ha OH 
 
 Distillate 
 
 Ether Extract of Residue 
 
 Standard Ha OH to 
 
 Standard Ha OH to 
 
 neutralize 
 
 Saponify 
 
 neutralize 
 
 Saponify 
 
 Hone 
 
 93. 8 
 
 Lost 
 
 ' Lost 
 
 One gram of potassium ethyl malonate and one gram of malonic 
 ester and 3 cc of boiled enzyme were dissolved in HaO and dis- 
 tilled in vacuo. The residue was acidified and extracted with 
 
 ether . 
 
 1 gram of potassium ethyl malonate is equivalent to 58.8 cc 
 0,09872 H Ha OH 
 
 1 gram of malonic ester is equivalent to 150 cc of 0 . 09872 N HaO?) 
 
 Distillate 
 
 Ether Extract of Residue 
 
 Standard Ha OH to 
 
 Standard Ha OH to 
 
 neutralize 
 
 Saponify 
 
 neutralize 
 
 Saponify 
 
 Hone 
 
 144.0 
 
 4 cc 
 
 32.0 
 

 - 
 
 1 1 
 
 
 
 
 
 
 . 
 
 
 
 
 
 
 
 
 
 
 
 
 . 
 
 
 
 
 
 
 
 
 
 
 
 
 
 * 
 
- 31 - 
 
 table XV 
 
 Isolation of products of hydrolysis of diethyl malonate and 
 diethyl succinate hy distillation in vacuo and ether extraction 
 of acidified residue. Duration of hydrolysis 3 ° days. O.Q9872 
 
 IT ITaOH was used for titration and saponification. 
 
 Standard 
 ITaOH re- 
 quired for 
 complete 
 sap on if i ca- 
 tion 
 
 Standard 
 NaOH ac- 
 tual lv 
 required 
 
 Diethyl 
 ester in 
 equiva- 
 lents of 
 
 0.09872 
 
 IT ITaOH 
 
 Ether Extract of 
 Ac id if i ed He s idue 
 
 Remarks 
 
 Standard 
 ITaOH to 
 neutral- 
 
 ire . 
 
 Standard 
 ITaOH to 
 Baponi— 
 
 L_£sl. , , 
 
 510 
 
 300 
 
 4.0 
 
 8.0 
 
 26.0 
 
 4 cc diethyl 
 malonate 
 neutral ized 
 daily 
 
 510 
 
 120 
 
 93.0 
 
 Undetermined 
 
 4 cc diethyl 
 malonate 
 H 2 O added 
 daily to keep 
 volume equal 
 to neutraliz- 
 ed mixture 
 neutralized 
 a t end 
 
 510 
 
 340 
 
 12.0 
 
 2 0.0 
 
 30. c 
 
 4 cc diethyl 
 malonate used 
 
 5 cc lipase 
 extract added 
 when equili- 
 brium reached 
 neutralized 
 daily 
 
 463 
 
 300 
 
 3.0 
 
 128.0 
 
 102.0 
 
 4 cc diethyl 
 succinate 
 neutral ized 
 daily 
 
 4^3 
 
 230 
 
 66 . 0 
 
 Undet g 
 
 s mined 
 
 4 cc diethyl 
 succinate 
 F 2 O added 
 daily to keep 
 volume equal 
 'gg that of 
 neutralized 
 mixture .neu- 
 tralized at 
 end . 
 

- 32 - 
 
 V DULMARY 
 
 I«A study of the hydrolysis of the diethyl est 
 the dicarboxylic acids 'ey liver 1 ij: ' 
 
 of hydrolysis changes with a., increase, in the molecular weight 
 of bhe ester. Under the sane experimental conditions lipase 
 app n\ removed both ethyl groups from diethyl adipate an 
 diethyl glutarate while the hydrolysis of diethyl r.aicnat e and 
 of diethyl succinate progressed only half way to completion. 
 
 II. * Crystals of pure adipic acid and no saponifiable 
 material were obtained from the hydrolysis of diethyl adipate 
 cy liver lipase when the acid produced by the reaction was neu- 
 tralized daily. 
 
 III. men the hydrolysis of diethyl adipate by liver 
 lipase was stopped half way in its progress, crystals of pure 
 adipic acid were obtained as one of the products of the hydro— 
 
 lysis* 
 
 J ' v * ; - n cases when 4 cc of ester was incubated with 
 liver lipase for a period of time the hydrolysis progressed 
 farther wren me acid, produced by the reaction was neutralized 
 
 daily . 
 
 ■ . u all cases when 4 cc of ester was incubated with 
 liver lipase over a period of time and the acid produced by the 
 reaction wcm not neutralized daily the extent of the hydrolysis 
 was greater when HsO was added daily than under otherwise simi- 
 lar conditions. 
 
 VI. The addition of 5 cc of enzyme to a reaction mixture 
 
. 
 
 
 
 
 
- 33 - 
 
 which had apparently reached an equilibrium had very little ef- 
 fect on the extension of the hydrolysis* 
 
 VII*With the exception of the experiments with diethyl 
 adipate complete recovery of the products of hydrolysis - as not 
 attained however, while the results of the methods employed in 
 general point to the conclusions of Lewis and Christman regard- 
 ing the hydrolysis of diethyl maloriate and diethyl succinate 
 they also open up various possibilities of research.