I. Studies on Mixed Anhydride Systems. II. Studies of the Alpha-Chymotrypsin Catalyzed Hydrolysis of Specific Substrates Citation Mower, Howard Frederick (1956) I. Studies on Mixed Anhydride Systems. II. Studies of the Alpha-Chymotrypsin Catalyzed Hydrolysis of Specific Substrates. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/031V-1769. https://resolver.caltech.edu/CaltechETD:etd-06162004-132607 Abstract The synthesis and purification of p-nitrobenzoic-acetic mixed anhydride, p-methoxybenzoic-acetic mixed anhydride and benzoic-acetic mixed anhydride is described. The thermodynamic constants for the disproportionation reaction have been determined. A mechanism for this reaction is discussed. Measurements of the interaction of acetic and benzoic acids with the acetic-benzoic anhydride system have been made. The cleavage of the mixed anhydrides with nucleophilic agents is described. The synthesis of the following compounds is described: N-trimethylacetyl-L-tyrosinhydrazide, N-dichloroacetyl-L-tyrosinhydrazide, N-formyl-L-tyrosinhydrazide, N-trichloroacetyl-L-tyrosinamide and N-acetyl-L-cysteic acid carboxylamide. The kinetic constants for the [alpha] chymotrypsin hydrolysis of these substances are reported. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Chemistry and Chemical Engineering) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Minor Option: Chemical Engineering Thesis Availability: Public (worldwide access) Research Advisor(s): Niemann, Carl G. Thesis Committee: Unknown, Unknown Defense Date: 1 January 1956 Record Number: CaltechETD:etd-06162004-132607 Persistent URL: https://resolver.caltech.edu/CaltechETD:etd-06162004-132607 DOI: 10.7907/031V-1769 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 2615 Collection: CaltechTHESIS Deposited By: Imported from ETD-db Deposited On: 17 Jun 2004 Last Modified: 13 Jul 2023 22:58 Thesis Files Preview PDF (Mower_hf_1956.pdf) - Final Version See Usage Policy. 8MB Repository Staff Only: item control page

‘I. STUDIES ON MIXED ANHYDRIDE SYSTEMS II, . STUDIES OF THE ALPHA CHYMOTRYPSIN CATALYZED HYDROLYSIS | OF SPECIFIC SUBSTRATES Thesis by Howard Frederick Mower In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 1956 ACKNOWLEDGMENTS it is a great pleasure to acknowledge the interest, encouragement and guidance of Professor Carl G. Niemann. His counsel throughout my graduate studies was greatly appreciated. My associates in the laboratory were of great help not only because of their cheery companionship but also through their many valuable suggestions. Throughout my graduate residence I have been continually grateful for the support given me by the California Institute of Technology through Graduate Teaching Assistantships and for the receipt of a Fellowship from the Shell O11 Company. The receipt of several Summer Grants from E. I. du Pont de Nemours is also gratefully acknowledged. I also wish to express my gratitude to my Mother, Mrs. Alice H. Mower, and to my fiancee, Miss Nancy C. Alpert, for their help, understanding and inspiration. ABSTRACT The synthesis and purification of p-nitrobenzoic-acetic mixed anhydride, p-methoxybenzoic-acetic mixed anhydride and benzoic-acetic mixed anhydride is described, The thermo- dynamic constants for the disproportionation reaction have been determined. A mechanism for this reaction is discussed, Measurements of the interaction of acetic and benzoic acids with the acetic-benzoic anhydride system have been made, The cleavage of the mixed anhydrides with nucleophilic agents is described. The synthesis of the following compounds is described: N-trimethylacetyl-L-tyrosinhydrazide, N-dichloroacety1-L- tyrosinhydrazide, N-formyl-L-tyrosinhydrazide, N-trichloro- acety1-L-tyros inamide and N-acetyl-L-cysteic acid carboxyl- amide. The kinetic constants for the & chymotrypsin hydrolysis of these substances are reported. I IL Iil TABLE OF CONTENTS PART I. Studies On Mixed Anhydride Systems Page Introduction . .. 1. 6. 1 2 6 ee ew ww Results and Discussion A. The Disproportionation Reaction B. The Kinetics of Disproportionation ... C. The Interaction of Acetic and Benzoic Acids with the Benzoic-Acetic Anhydride System . 6. 6. 6 ee ew ew ew ee ee D. Nucleophilic Cleavage of the Mixed Anhydrides .... 6. 2 1 we se ew ee ew Experimental Section A. Preparation of Starting Materials .... B. Demonstration of the Interconversion of Symmetric and Mixed Anhydrides ..... C. Analytical Techniques .........-. D. Estimation of Probable Error ...... E. References ... 2... 6 6 ee we ee F., Experimental Data | a. Benzoic-Acetic Anhydride System . Summary of Beer's Law Data ... - Beer'ts Law Data for Acetic Anhydride ..... 2. 1. 2. ee ee NO cee Beer's Law Data for Acetic Acid . Beer's Law Data for Benzoic Acid e 7 s s . se 2 ° e ° s ca * s 3. Beer's Law Data for Benzoic Anhydride ..... o 8 4, Beer's Law Data for Benzoic- acetic Mixed Anhydride ... . 5 6 * @ 34 43 46 73 p-Methoxybenzoic-Acetic Anhydride System 1. Summary of Beer's Law Data . . 2. Beer's Law Data for p- Methoxybenzoic Anhydride .. 3. Beer's Law Data for p-Methoxybenzoic— acetic Mixed Anhydride 4. Beer's Law Data for Acetic Anhydride. p-Nitrobenzoic-Acetic Anhydride System 1. Summary of Beer's Law Data .. 2. Beer's Law Data for p-Nitrobenzoic. Anhydride .... . 3. Beer's Law Data for p-Nitrobenzoic- acetic Mixed Anhydride .. 4. Beer's Law Data for Acetic Anhydride. Bquilibrium Mixtures 1. Benzoic-Acetic Anhydride System . 2. p-Methoxybenzoic-Acetic Anhydride System .. . 3. p-Nitrobenzoic-Acetic Anhydride System . e * e ° e . ° s s e ° . e ° Kinetics of Disproportionation 1. Rate of Disproportionation of p- Methoxybenzoic-acetic Mixed Anhydride 2. Rate of Disproportionation of Benzoic-acetic Mixed Anhydride N-Acyl~p-Toluidine System summary of Beer's Law Data ... Beer's Law Data for N-BenZoy)-p- toluidine .. os ee Beer's Law Data for N- Acetyl- ~p- toluidine . MR p-toluidine. . Beer's Law Data for N-b- Nutrobenzoy?)- p-toluidine ..... oo : wm FF WwW Cleavage of Mixed Anhydrides with p- Toluidine ...... see es Apparent First Order Rate Constants for the Hydrolysis of the Three Mixed Anhydrides . . 1. 6 6 ew ew ee ee we Beer's Law Data for N- (p-Methoxybenzgyl )- Page 97 99 101 103 105 107 109 lil 112 121 125 129 133 140 142 144 147 148 149 155 PART II, Studies Of the Alpha-Chymotrypsin Catalyzed Hydrolysis of Specific Substrates T Results and Discussion A. B. Kinetics of the Hydrolysis of Three N-Acyl- The Ammonium Salt of N-Acetyl-L-cysteic Acid Carboxylamide as a Substrate ...., TI Experimental Section A. B. APPENDIX The Synthesis of N-Trichloroacetyl-L-tyrosinamide Synthesis of the N-Acyl-L-tyrosinhydrazides . Synthesis of the Ammonium Salt of N- Acety1- -L- eysteic Acid Carboxylamide .... oe Analytical Procedure . .. . «6 «© « «© © « « ReferenceS . 2. . 6 © © © © © © w eh we ew Experimental Data 1. rm Hydrolysis of N- Trimethylacety)- he tyrosinhydrazide ... we Hydrolysis of N-Formyl- “Le tyrosin- hydrazide ... coe Hydrolysis of N- Dichlozoacety1- “Le syrosinhydrazide .. Hydrolysis of N-Acetyl- -L- cysteic “Acid Carboxylamide .. . Inhibitor Studies of N- “Acetyl- L- cysteic Acid Carboxylamide . 8 Studies on Effect of Added Sodium Sulfate . 2. 2. 2 6 6 6 6 we ew ew we et ee Page L57 162 164 L170 173 LTT 178 209 210 215 218 ‘PART I, STUDIES OF MIXB#D ANHYDRIDE SYSTEMS INTRODUCTION Mixed anhydrides I, formed by the loss of water from two different carboxylic acids, have been known or postu- — REC-OH RC, | + —— > 3 J (x) +H,0 R-c-oH a lated for a long time. Until the advent of high vacuum techniques the liquid members of this class of compounds could not be prepared in a pure state. Any attempt at fractionation of these compounds at temperatures above one hundred degrees centigrade resulted in the production of a distillate, whose boiling range was continuous from the boiling point of one symmetrical anhydride to the boiling point of the other symmetrical anhydride. Therefore, it was not certain whether this material represented a new compound or a mixture of equal portions of the two symme- trical anhydrides. This point was partially clarified by Behal, ‘1) who prepared several crystalline solid mixed anhydrides and showed them to have discrete properties “distinct from either of the symmetrical anhydrides. Other evidence for the existence of these Substances was ootained by Kilpatrick, (2) who Showed that acetic-proponic mixed anhydride hydrolyzed at a rate which was intermediate be- -2- tween the rate of hydrolysis of acetic and proponic anhy- drides. The reaction accurately followed first order kinetics throughout its course, while an equimolar mixture of acetic and proponic anhydrides gave kinetics which was not homogeneously first order. (3) Except for their relationship to the Perkin synthesis (4) and certain acylation reactions, interest in these sub- stances has lagged until the last few years. Recent develop- ments in the field of peptide synthesis have greatly increased the importance of these compounds and have stimulated efforts to understand their properties, (5) (6) Wieland and Vaughn have developed new peptide syntheses based upon the conversion of an acylated amino acid or peptide into a mixed anhydride, The mixed anhydride is then allowed to react with the free amino group of another amino acid to form a peptide bond. It was soon found very convenient to form the acylated amino acid mixed anhydride with an alkyl chlorocarbonate. 9 R-CcH-C-OH 1 P _! _ oe ‘nu R arc O-c-OR Rien Cnr fo { > R SS vo) EtsN Ru bxo NHR Ree + On The rate and the magnitude of equilibrium constant of the disproportionation reaction and the products obtained by heterolytic cleavage with nucleophilic agents are the proper- -3- ties of a mixed anhydride which one is most interested in being able to predict. Mixed anhydrides all show a tendency to disporportionate into the two symmetrical anhydrides 9 5 ff 9 Roc ——= RG + R Sy ad t P RE é a ° It is possible to show that this reaction is reversible and that an equilibrium state is reached. However, quantitative measurements of this equilibrium mixture were not possible until the advent of infrared spectophotometric techniques, Since it was not possible to devise a chemical test which would quantitatively distinguish between the mixed anhydride and an equal molar mixture of the two symmetrical anhydrides. Brown and Trotter?) were the first to carry out quanti- tative measurements on a mixed anhydride system. They showed that if a mixture of acetic and butyric anhydrides is allowed to stand and its infrared spectrum examined as a function of time, the spectrum changes from an additive mix- ture of acetic and butyric anhydrides to that of a mixture of the two symmetrical anhydrides and a third substance. Dur- ing this change a prominent new peak appears in the 8-9 micron region. [the region of the C-0O-C stretching frequency of anhydrides]. The appearance of this peak is accompanied by a corresponding decrease in the peaks associated with -he either symmetrical anhydride. Therefore, the new peak is assigned the C-0-C stretching frequency of acetic-butyric mixed anhydride. It was shown that after several hours the mixed anhydride peak attains constant dimensions and that it is invariant with changes in temperature. From this fact it was determined that the change in enthalpy for the disproportionation reaction was zero and that only entropy considerations should affect the over-all free - energy change. Thus the disproportionation reaction seems to be Similar to certain processes of ester interchange, of metal alkyl interchange and of the distribution of halogens between glycol dihalides., These reactions were first recog- nized by Calingaert (8) and called redistribution reactions. they are characterized by a statistical distribution of organic radicals about a central moiety. If the mixed anhydride disproportionation reaction is a redistribution reaction, then the equilibrium constant should be four. This hypothesis was tested and found to be consistent with the data obtained for the acetic-butyric anhydride system, Thus Brown and Trotter were able to measure the equili- brium constant for the acetic-butyric anhydride system with- out ever having isolated or purified the mixed anhydride. In like manner, they were able to measure the rate of dis- proportionation of the acetic-butyric mixed anhydride and found the second order rate constant to be “~A = 4.105 Liters moles~/ min. > at 398 K -5~ One of the most important reactions of mixed anhydrides is their cleavage by nucleophilic agents. This reaction constitutes the basis for many acylation reactions. By the work of oral?) it has been shown that for most mixed anhydrides this reaction can be considered as an Sye attack by the nucleophilic reagent upon the carbonyl carbon- atom which carries the greatest positive charge and which is the least sterically hindered for such an attack. Often it is found that the factors which increase the positive charge of the carbonyl carbon also increase its steric shielding. Thus in the series: acetic-monochloroacetic, acetic-dichloroacetic, acetic-trichloroacetic mixed anhydride, it is the monochloroacetic mixed anhydride which has the best combination of characteristics to give the highest yield of & haloacylation. Often the effective steric size of substituents on the & carbon will vary with the reaction medium. If there is a delicate balance between the suscepti- bility of the two carbonyls to nucleophilic attack, then striking variations in product composition can be obtained by changing the reaction medium. Thus, in benzene, chloro- acetic-acetic mixed anhydride gives six times as much chlioro- acetylation as acetylation, but in aqueous solvents two simes as much acety.avion as chloroacetylation is obtained. In the absence of strongly protolytiec solvents it is believed that acylium ions do not play an important part in the reactions of carooxylic anhydrides. However, Tedder _6- 1 et ar.' °) have shown that trifluoroacetic mixed anhydrides seem to constitute an exception to this rule. In this case some reactions seem to proceed by a preliminary ionization into trifluoroacetate and acylium ions. STUDIES ON SYSTEMS OF PARA SUBSTITUTED BENZOIC-ACETIC MIXED ANHYDRIDES Aim and Scope of the Studies The purpose of this work is to study the properties of three para substituted benzoic-acetic mixed anhydride sys- tems; the benzoic-acetic, the p-nitrobenzoic-acetic, and the p-methoxybenzoic-acetic mixed anhydride systems. The investigation of the disproportionation reaction and the heteroiytic cleavage with nucleophilic reagents was under- taken to learn whether the behavior of these substances was compatible with the ideas that were advanced in the introduc- tion. In addition these studies were extended to include the interaction of acetic and benzoic acids with the benzoic- acetic mixed anhydride system. It is hoped that the results of this work will be of some help in the design of future synthetic procedures in the field of amino acid chemistry. The Disproportionation Reaction Introduction The work of Brown and Trotter(7) Suggested that the nature of the disproportionation reaction might be relatively independent of the structure of the mixed anhydride and re- -gardless of the character of the organic rest attached to the carbonyl carvon, the equilibrium constant would be -8- four and the temperature independent. To test this hypothesis, benzoic-acetic, p-nitrobenzoic- acetic, and p-methoxybenzoic-acetic mixed anhydrides were prepared in a pure state. These compounds differ from one another in regard to the positive character of the phenyl ‘carbonyl carbon atom and should demonstrate any effect “which arises from this cause. It is fortunate that these mixed anhydrides are of sufficient stability to allow their purification. This allows one to remove any doubt as to the interpretation of the change in the infrared spectrum that occurs when the two symmetrical anhydrides are heated to- gether. The presence of the pure mixed anhydride allows one to approach the equilibrium point from two directions and to establish its value with greater certainty. Results and Discussion By use of the infrared spectrophotometric tecnnique described in the experimental section, the concentration of the three species comprising an equilibrium mixture can be measured and an equilibrium constant, K, determined. The results are shown in Tables 1, 2, and 3. co K= (mixed anhydride) (Acetic anhyd) (Phenyl anhyd) -9- Table 1 Benzoic-~acetic Anhydride System Equilibrium constant derived from mixtures with initial compositions of: Acid and Temperature Mixed Anhydride Symmetrical Anhydrides an Anhyd 70°C 1.07 1.04 75°C -~ -- 100°C 1.85 1.98 150°C 4.17 431 A plot of the ln K vs. ris shown in Figure i. 1.08 1,25 3.12 2.00 494 3.74 From this plot, the change in enthalpy for the disproportionation re- action was determined as M@® H = 5.0 Keal. Table 2 p-Methoxybenzoic-acetic Anhydride System Equilibrium constant derived from mixtures with initial compositions of: Temperature Mixed Anhydride Symmetrical Anhydrides 75° 2.31 2.27 2.25 100°C A, 25 4.22 4,69 105°C 9.13 11.93 A plot of in K vs. i is shown in Figure 2. From this plot, T the change in enthalpy for the disproportionation reaction was determined as 4H = 5,8 Keal. ink 1.5 0.7 0.6 0.5 0.4 0.3 0.2 -1lO- \ Figure 1 \ Plot of In K vs. # for Benzoic-Acetic . Anhydride System 2.0 2.1 2.2 2. i 2, 4 2.5 2, 6 2, 7 2, 3 2.¢ 3.0x1073 ~Li- Figure 2 Plot of in K vs. . for p-Methoxybenzoic-Acetic Anhydride System dn K | \ 2 . 6 ‘me \ - 2,4-— \. 2,2- \ 2.0L 1.8}- : . 1.6+— 1A | = 1.2)— L,Or— ore 0.8 0.6. O.,4,— rtirtp.ty ty typ tp tp ty ft cpl tl | . 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.6 3.0x1073 ae -12- Table 3 p-Nitrobenzoic-acetic Anhydride System Equilibrium constant derived from mixtures with initial compositions of: Temperature Mixed Anhydride Symmetrical Anhydrides 75°C —s—=«<iO:T 1.20 100°c 1.29. 1.32 7 9 1.38 150°C 1.70 1.78 A plot of In K vs. r is shown in Figure 3. From this plot the change in enthalpy for the disproportionation reaction was determined as A H = 1.04 Keal. The above results show that the equilibrium constants for the disproportionation reaction of each of the anhydride systems have a decided temperature dependency and hence are “in marked contrast to the aliphatic mixed anhydrides. It appears at the present time that there is no simple explanation either of the differences of the substituted benzoic-acetic mixed anhydrides between one another or of the differences between the substituted penzoic-acetic mixed anhydrides and the aliphatic mixed anhydrides. Hammett?1) has set forth the following relation which describes the change in free energy which takes place during a reaction: AF =-RTInk= AEp+AE,~ RTn SF ~13- Figure 3 Plot of In K vs. F for p-Nitrobenzoic- \ Acetic Mixed Anhydride System in K \ 0.6 0.5 0.4 0.3 0,1 N ae 0 a vty tartar titi ts tp ta lo rt a 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0x1073 1 T -14- The term A Ep is the difference in potential energy between the reactants and the products, AE, is the dif- ference in zero point energies of the various reaction species present and fy and t. are the partition functions of the products and reactants. The term AE,is the energy ‘change which would occur if the reaction could take place so that none of the reactive species would possess the energy of motion that is associated with the various. vibra- tions and rotations that the molecules are able to undergo. The logarithmic term and the quantity AE, are concerned then, with the kinds of rotational and vibrational motion that the molecules can execute and with the various energy-level spacings of the reactants and products. The term Ap is the quantity which is influenced by changes in resonance energy, electron displacements, dipole interactions, etc. Unless the kinetic terms of the Hammett equation can be cal~ culated or shown to be insignificant, it is not possible to discuss the effectof changes in structure upon reaction rate, equilibrium constant or heat of reaction entirely in terms of changes in bond strength, resonance energy, dipole interactions, etc. If the products and reactants of a reaction are so Similar that the energy level spacings are nearly the same then, xv An St +O > ~15- and AF=AE, > Since AVYand AP are small; AF=A4H AS=0 Reactions between compounds which satisfy these condi- tions can be described by the use of the Hammett functions P and @. Meta and para substituted benzene derivatives, in which the reaction takes place at a reactive center ad- jacent to the benzene ring, constitute the classical examples in which this type of analysis is possible. Aliphatic com- pounds in general do not display these properties. Table 4 shows the thermodynamic constants of the dis- proportionation reaction for the three anhydride systems. It is evident that AS is far from being zero and AF is not even of the same sign as AH, Figure 4 shows the logarithm of the equilibrium constant for the disproportionation reac- tion plotted against Hammett's constant for the para nitro, para methoxy and the unsubstituted benzoic-acetic mixed anhydride. It is seen that the deviations froma linear relationship are rather large. Table 4 Thermodynamic Constants for the Disproportionation Reaction of the Para Substituted Benzoic-acetic Mixed Anhydrides at 100° C. A F(cal) BH(cal) AS§cali/Pc p Nitro -212 1,252 3.4 p Methoxy -1.088 5,800 18, p Hydrogen -480 5,000 14.7 ~16- Figure 4 Plot of log K vs. Hammett's Function 0 for the Three Para Substituted Benzoic- log K acetic Mixed Anhydrides. 0.7 log K Determined at 100° ¢, ® 0 CH 0.6 -~ 0.5 -— O.4 -— 0.3 -- *H O.e te NO, ° OL }-— -0.3. -0.2 -O.1 0.0 40.1 0.2 0.3 0.4 0.5 0.6 0.7 0 -17- It was hoped that because the number and types of bonds in the mixed anhydride as compared to the symmetrical anhydrides are so nearly identical, this system could be analyzed in terms of changes in potential energy alone. The failure for this partly lies in the importance of the acetyl carbonyl group, which is removed from the phenyl group by a carbon and oxygen atom, as a reactive center in the disproportionation reaction. Although it is probably not realistic to describe the differences in the thermodynamic characteristics of the mixed anhydrides in terms of changes in potential energy alone, it should be pointed out that a certain amount of correla- tion can be obtained by discussing these substances in terms of classical resonance structures. It is noted that in each case the sign of the AH term is positive and hence the mixed anhydride is less stable than the symmetrical anhydrides. Proceeding from the para methoxy compound to the para nitro compound the differences in AH become smaller. Weilana®?) has calculated the resonance energy for acetic anhydride as 41 Keal/mole and a value of 29 Kceal/mole has been estimated by Franklin, (23) Wieland suggests that this resonance energy is due to the following interactions: R-GO R-C-O™ R-c=0 Oo <> Yom <> / Pom po R-C=O R-C=0 R-C-O0® ~18- It is to be expected that substituents in the para position of the symmetrical benzoic anhydrides might well affect the importance of structures such as III. c~) re) Pr x46 cOx —At Structure III involves the creation of a charge on the central oxygen atom, and this charge is adjacent to a car- bonyl carbon which already carries a partial positive charge. This situation tends to decrease the stability of III. If the para substituent X is a methoxyl group, then the partial positive charge on the carbonyl carbon is diminished and the resonance form III is of increased stability. A para nitro group exerts the opposite effect, and with an increased charge on the carbonyl carbons the contribution of III to the structure of p-nitrobenzoic anhydride is smaller, At the same time the effect of the para nitro eroup is to diminish the stability of only one of the corresponding structures (IV - A) in the mixed anhydride. . | Oo Oo , 9H aie x O-CO=CCHs > X-QO-c=O-C-CHy Ty-A Tv-B -19- The effect of the substitution of a para nitro group is to decrease the energy difference between the symmetri- cal and the mixed anhydride by decreasing the resonance energy of the symmetrical anhydride to a greater extent than it decreases the resonance energy of the corresponding “mixed anhydride and thus in a rough way to decrease the value of AH. | In the mixed anhydride the phenyl groun may interact with the resonating system of the carbonyl groups so as to destroy the equivalence of the two structures corresponding to III. This would generally tend to decrease the stability of the mixed anhydrides relative to the symmetrical anhydrides. Kinetics of the Disproportionation Reaction Using the infrared spectrophotometric techniques de- scribed in the experimental section, it was possible to follow the disproportionation of a mixed anhydride into the symmetrical anhydrides and to examine the kinetic conse- quences of this reaction. The p-nitrobenZzoic-acetic mixed anhydride dispropor- tionated at a rate that was considerably faster than either the p-methoxybenzoic-acetic or the benzoic-acetic mixed anhydrides. Because of its rather low solubility in chloro- form, this was not a convenient case to study. With p-methoxybenzoic-acetic and benzoic-acetic mixed anhydrides the disproportionation reaction displayed kinetics -20- which could be unambiguously determined as a second order with respect to the mixed anhydride. See Figure 5A and B. Only the initial 20% of the reaction was followed, as at this point the back reaction became important and the apparent rate constant continuously decreased to zero... Second Order Rate Constants for the Disproportionation of p-Methoxybenzoic-acetic Mixed Anhydride A. Reaction followed by the disappearance of the mixed anhydride at 100°C ky = 1.29 x 10°39 moles”? liters min.~4 1.38 x 10-3 it 1 " B. Reaction followed by the appearance of p-methoxybenzoic anhydride at 100°C k, = 1.34 x 1073 moles7! liters min.7? "! " © 4.60 x 1073 1" Second Order Rate Constants for the Disproportionation of Benzoic-acetic Mixed Anhydride Reaction followed by the disappearance of the mixed anhydride at 100°C ky = 2.27 x 1073 moles“? liters min.~+ 2,82 x 1073" noon 2.43 x 1073 |" " n 2.16 x 103" " " From the additional data presented in the experimental section, it is seen that the reaction is homogeneous, i.e., powdered glass does not affect the rate constant. The addi- tion of sodium acetate increases the rate somewhat (p. 137). Ot. GE O€ G2 Od GT OT G | | v ' { | | ] | ] Td l ] | 49 ” ae ~ | 8 o. a a —_ a © a — —| ot a a fat e. t Lao = _ ot a a a a a — tT (OTXD) 301 out. "SA q- (dora BagU80U09 ) uoTQoBvey uoTZeUCT{ZUOdOAdSTq AoJ ZOTZ dapdo puovas WS amn3Ty | On GE SH OS -2e- aUTL “SA (OTXD) BOT uoTzovay uoTyeuoT{AOdoadstq Jos 4OTd AePIQ ASAT (uFH)s of Se Od St | | ee ee ee OT G Q —~2°0 —E€°O —=3S°O (OTXO) SCT dG aan3ta -23- (14) This is easily explained in terms of the work of Evans, who showed that acetate ion labeled with ct very rapidly exchanges with acetic anhydride. Acetic or benzoic acid also increases the rate somewhat. This is understandable in view of the work in the next section. The evidence set forth effectively excludes from con- sideration of the mechanism for the disproportionation reac- tion any scheme which involves as a rate determining step the production of ions as suggested below. 2.0 CHE ° Py 0 —> CHrGO? + Rather it is believed that the second order reaction kinetics represent a manifestation of a true bimolecular mechanism. It is suggested that the approach of the two reacting molecules is facilitated by the dipole-dipole interaction of the carbonyl groups and that the "reactive intermediate" has the structure shown below. C Hz FAs CH CH “<Q OF oy? rr3 CH3 CH = Sr. | 3 a a O= O= Pr. YO 7! \) CzO * x x + Ose-%Wc-0 x The breakdown of the active intermediate is favored by a concerted shift of carbon-oxygen bonds throughout the pseudo six-membered ring. The initial orientation of the reacting molecules is important and determines whether the combination could lead to the same or different species. Because of steric con-~ Siderations, most reactions which are capable of resulting in a disproportionation probably proceed through an orienta- tion of the activated complex in which the initial contact is formed by the carbonyl functions which have the methyl groups attached (see p. 29 ). ~25- CH en 1) CH; O-E---O-CM Po fom 43) x x . After the initial interaction of the carbonyl functions to which the methyl groups are attached, one can imagine that the facility with which the activated complex breaks down to give the disproportionated products is governed by the ease with which the carbon-oxygen bond two-three is formed and the carbon-oxygen bond three-four is broken. It is suggested that the mechanism of the disproportionation re- action is such that the rate determining step is the nucleo- philic attack of the oxygen atom number two upon the car- bonyl carbon atom number three. It is expected that the greater the susceptibility of this carbonyl atom to nuclé.o- philic attack the faster will the rate of disproportionation proceed. The susceptibility of the carbonyl carbon atom to this type of attack will be profoundly affected by the nature of the para substituent X, If this substituent is a nitro group, the nucleophilic attack would be expected to proceed more readily than if the substituent is a methoxyl group. The experimental results confirm the prediction, formu- lated on the basis of this hypothesis, that the rate of disproportionation of the p-nitrobenzoic-acetic mixed anhy- -26~ dride.is greater than the rate of disproportionation of p-methoxybenzoic-acetic mixed anhydride, and that the rate of disproportionation of the benzoic-acetic mixed anhydride has a value intermediate between these two, Brown and trotter(?) found that the rate of dispropor- tionation of butyric-acetic mixed anhydride was one thousand times greater than the rate of disproportionation of benzoic- acetic mixed anhydride, The Interaction of Acetic and Benzoic Acids with the Benzoic- Acetic Anhydride System. By extending the infrared spectrophotometric techniques used to measure the anhydride systems to a five component mixture, it is possible to quantitatively measure the inter- action of acetic or benzoic acids with the benzoic~-acetic anhydride system. The para-methoxybenzoic or para-nitroben- ZOolc-acetic anhydride systems were not suitable for this study, as para-nitrobenzoiec and para-methoxybenzcic acids are not soluble in chloroform. in addition to the equilibrium involving only the anhy- drides, the system can be described by defining two other equilibrium constants corresponding to reactions I and II. om = clon + oO P CH; C=0 | gre O [ HAc| £ 52,0] [Pep] [HP] K (Aco 3) = -27- oe) y Cis ¢ Sg C Oyen Sp = csbors CIS, / CHC Clg C © LWA abe] K (Acvp1L) = [Ac,O] LHe] Table 5 Composition of Temperature K (anhyd) K (acid-I) K (acid-II) Initial Solution 75°C 1.25 1.58 1.97 acetic anhyd, ben- zoic anhyd, acetic acid Oo 75°C 1.08 3.36 3.61 benzoic-acetic mixed anhyd, ben- ZOic acid 00 1.85 2.04 penzoic-acetic mixed anhyd, ben- zoic acid Mm 100°C fe) 100 C 3.12 2.23 6.95 benzoic anhyd acetic acid 150°C 3.74 1.27 LTS benzoic-acetic mixed anhyd, acetic acid 150°C bolt 1.29 6.33 acetic anhyd, benzoic acid Unfortunately the accuracy of the analytical technique does not permit, with this complicated mixture, a very ac- curate determination of the equilibrium constants associated “My _28- with the interaction of the acids with the anhydrides. It is thus not vossible to describe the details of the mechanism either in terms of a redistribution reaction or in terms of the disproportionation reaction of the para substituted benzoic-acetic mixed anhydrides. If the acid interaction “followed the principles of a redistribution reaction, then the values of the first acid equilibrium constant should be one half and those for the secone acid equilibrium constant should be two. A source of ambiguity is the uncertainty over which molecular species of the acid is able to react with the anhy- drides. Recent work’ 12) has indicated that in the concentra- tion range in which these measurements were made acetic acid is almost entirely converted to the dimeric form. It is possible to write a mechanism for the reaction of the mono- meric acid with an anhydride which is formally quite similar to that proposed for the disproportionation reaction. /CH ey _F Hs, < OEM — ae } Om Hes + No ae ) From this point of view it does not appear likely that the dimeric form of the acid would react as readily as the monomeric species. if the monomeric form of the acid is the only one which reacts, then the true acid equilibrium -29- constants will be proportional to the square roots of the observed (or dimeric) acid concentrations. If the values for K (acid-I) and K (acid-II) are corrected by using the square roots of the observed acid concentration, no improve- ment in the consistency of the results is observed. To ‘settle this point a more sensitive analytical procedure will have to be employed. The Nucleophilic Cleavage of the Benzoic-acetic, p-Methoxy- benzoic-acetic, and p-Nitrobenzoic-acetic Mixed Anhydrides. In this section attention is directed towards the reaction of a mixed anhydride with nucleophilic agents in general and amines (p-toluidine) in particular. In the experimental section an analytical scheme is presented which enables one to determine the product composition of the reaction between a mixed anhydride and p-toluidine. From this procedure a quantity called the acetylation ratio is determined. It is defined as the amount of N-acetyl-p- toluidine found in the gross product divided by the amount of N-(p-substituted penzoyl)-p-toluidine found in the gross product. -30- Table 6 Acetylation Ratio Reaction Benzoic-acetic p-Methoxybenzoic- p-Nitrobenzoic- Medium Mixed Anhya acetic Mixed Anhyd acetic Mixed Anhyd Benzene 43.9 102.4 2.7 50 why . Acetone- 37.7 140.7 4.7 ' Benzene Acetone 25.6 120.5 3.5 50 w/w Acetone- 42.5 93.8 3,3 Water It is evident that in this case the acetylation ratio is relatively independent of the polarity of the reaction solvent. This result can be expected when there are no substituents which could shield the carbonyl groups and whose steric size could be altered through changes-in the amount of their solvation. Thus one would not expect the results that go1a'?) obtained with a chloroacetic-acetic mixed anhydride whose acetylation ratio was highly dependent upon the polarity of the reaction mediun. All three of the para substituted benzoic-acetic mixea anhydrides yield a preponderant of N-acetyl-p-toluidine, demonstrating the steric effectiveness of the phenyl group in hindering the attack of p-toluidine at the carbonyl func- tion which is attached to the phenyl group. The acetylation ratio decreases as one passes from p-methoxybenzoic-acetic mixed anhydride through benzoic- -31- acetic mixed anhydride to the p-nitrobenzoic-acetic mixed anhydride. The marked difference between the p-nictro and p-methoxy compounds is probably due to the combination of mesomeric and inductive effects, which for the p-nitro group will increase the positive charge at the carbonyl carbon atom adjacent to the phenyl ring and for a p-methoxy group will decrease the charze. It is expected that as the charge on one of the carbonyl carbon atoms is increased by substi- cution in some part of the molecule, the nucleophilic at- tack will be increasingly favored at that position, and hence the acetylation ratio will change accordingly. In like manner it would be expected that the rates of hydrolysis of a mixed anhydride should be affected in a Similar fashion. The more the positive charge of the car- bonyl carbon atom adjacent to the phenyl ring is decreased by the action of substituents in the phenyl ring the slower the rate of hydrolysis. As the positive charge on the carbonyl group is increased, it is expected that the rate of hydrolysis will increase, since the nucleophilic attack would not tend to discriminate against one of the reactive centers. This prediction has been verified, as is seen in Taole 7, where apparent first order hydrolysis constants 16) for the mixed anhydrides are given. Berliner! also ob- tained similar resuits with the symmetrical para substituted anhydrides. -32- Table 6 Hydrolysis Constants for Para Substituted Benzoic-acetic Mixed Anhydrides in 50% w/w Tetrahydrofuran-water p-Nitrobenzoic- p-Methoxy-~- Benzoic-acetic acetic Mixed benzoic-acetic Mixed Anhyd Anhydride Mixed Anhyd ‘Apparent first _ _ . _ _ order k = 3.77x1075See > 23.65x107°See7> 1.42x107°See 7+ rate constant k -33- EXPERIMENTAL SECTION, PREPARATION OF STARTING MATERIALS Symmetrical Anhydrides A brief survey of available methods for the prepara- ‘tion of symmetrical anhydrides was made. At that time it was judged that the procedure suggested by Wheland!>?? was the most convenient and gave high yields consistent with purity. This method is based upon the reaction of a carboxylic acid with phosgene in the presence of a tertiary amine and in an inert solvent to form an unstable mixed anhydride of carbonic acid. The mixed anhydride then undergoes rapid dis- proportionation forming the symmetrical carboxylic anhydride and carbon dioxide. 0 0 0° 0 1 i ue oou ee 2R-C-OH + C1-C-Cl — R-C-0-C-0-CR + Et.NHC1 Et,N 3 0 0. O 0 il I (I R-C-O0-C-O-CR -——> R-C-O0-C-R + C05 1 \\ ) 0 It was found necessary to carry out the reaction at -10°C where yields of 80% were obtained. At one only 10% of the desired product was formed. -35- Procedure In the following manner benzoic anhydride, symmetrical p-methoxybenzoic, and symmetrical p-nitrobenzoic anhydrides were synthesized: One tenth mole of the desired acid was dissolved in 250 ml. of ary tetrahydrofuran.» The solution was placed in a 500 mil. three necked round bottomed flask equipped with a sealed mechanical stirrer and protected from the at- mosphere with a calcium chloride drying tube. Twenty-four one hundredths mole of dry triethylamine was added and the mixture cooled to -10°C in an ice salt bath. A solution of 0.11 mole of phosgene in 50 ml. tetrahydro- furan was prepared at room temperature and then cooled to -10°c. The cooled phosgene solution was slowly added by means of a dropping funnel to the stirred solution. of acid and triethylamine. The process of addition requires one half hour. The solid triethylamine hydrochloride was separated by rapid filtration and the solvent removed by distillation under reduced pressure in the presence of nitrogen. The results are shown in Table 7. In order to detect small quantities of the parent acid x - Tetrahydrofuran is preferred as a reaction solvent, since it dissolves larger quantities of the aromatic acids than do the other tow voiling inert solvents. It may be con- veniently dried by distillation over sodium wire but can- not be stored in the presence of sodium without further treatment with lithium aluminum hydride to avoid slow deterioration. -36- in a samole of anhydride, recourse can be made to spectro- photometric analysis in the infrared spectra. The carbonyl stretching frequencies in the neighborhood of 5.7-5.9 microns are used, Since they are very intense bands. The absorption bands of the anhydride are shifted 0.13 microns toward Shorter wave length relative to the corresponding bands of ‘the acid. Using this method it is possible to detect five parts in 1000 of acid in anhydride. The final criterion of purity of a sample of anhydride was the complete absence of any absorption oands of the acid in the spectrum of the anhydride. Table 7 Preparation of Symmetrical Anhydrides Wave length of Anhydride M.P. after First Final Characteristic Recrystallization Yield Final M.P. Adsorption Acid Anhyd. Benzoic 42 0-44..5 71% HY 5-45.06 5.86 5.78 p-Methoxy - benzoic 87-92 53% 95.5-96.2 5.87 5.74 p-Nitro- pvenzoic 188-190 48% 191.5-192.0 5.90 5.79 Recrystallization Solvent Benzoic Anhydride € Vol. Petroleum Ether . 1 Vol. Benzene p-Methoxybenzoic Carbon Tetrachloride p-Nitrobenzoic Purified Acetone -37- The acetic anhydride used in the study was purified by distillation over sodium wire and fractionated through 150 em. of helices in a Todd column. The middle third portion was retained. The material was stored over sodium wire. Each preparation was used for two weeks only, as acetic ‘anhydride seems to attack sodium wire slowly at room tempera- ture. Mixed Anhydrides (Para Substituted Benzoic-Acetic Mixed Anhydrides } An old method of obtaining mixed anhydrides involves the reaction between the sodium salt of one acid and the acid chloride of another, This method suffers from the fact that the sodium chloride forms a finely divided preci- pitate and a colloidal suspension usually results. Separa- tion of this reaction mixture usually involves lengthy filtration or contracted centrifugations, which result in lower yields. The method of choice is therefore the reac- tion of ketene and the desired aryl carboxylic acid. This results in the case of benzoic and p-methoxybenzZoic acids in rapid quantitative conversions to the para substituted benzoic-acetic anhydride. p-Nitrobenzoic acid, however, failed to react ina straight forward manner with ketene, and it was necessary to fall back upon the reaction between sodium p-nitro- benzoate and acetyl chloride. -38- Preparation of Mixed Anhydrides by Reaction of Aryl Carboxylic Acids and Ketvene One hundredth mole of the desired acid wasdissolved in 200 ml. of dry tetrahydrofuran contained in a 500 ml. Erlen- meyer flask. The flask was then placed on a magnetic stirrer and mildly agitated, while a slow steady stream of ketene “was passed into the solution. The reaction was followed by dipping a stirring rod into the solution and allowing the adhering solution to evaporate. As the reaction proceeded smaller quantities of solid remained on the stirring rod after the evaporation of the solvent, and at the end of the reaction only a heavy oil was produced. Near the end of the reaction the rate of addition of ketene was decreased and the solution closely observed. AS Soon as a slight brown color had appeared, the reaction was stopped. This brown color is due to a slightexcess of ketene in the solution, which is quickly converted into dark colored condensation products. The ketene was produced by the pyrolysis of acetone as described by Hurd.'17) ‘the time for addition of ketene was 45 minutes. The solvent was then removed from the reaction mixture by distillation under reduced pressure and the residue purified by fractionation or recrystallization. Yield after (B.P. 88-89°at Benzoic-acetiec Mixed Anhydride Fractionation 80%( 0.2 mm. M.P. 1.5°C o Nis= 1.5152 p-Methoxybenzoic-acetic Yield after M ixed Anhydride Recrystallization 75% ( from Benzene ( ren -39- Synthesis of p-Nitrobenzoic-acetic Mixed Anhydride When p-nitrobenzoic acid was treated in the above man- ner with ketene, only starting material could be obtained. Therefore, it was necessary to utilize the reaction between sodium p-nitrobenzoate and acetyl chloride to obtain the ‘desired product. The mixed anhydride is a relatively unstable substance. It reacts quickly with any moisture in its environment and upon heating is rapidly disproportionated to the symmetrical anhydrides. Therefore, in order to prepare pure samples of this anhydride, it is necessary to carry out the reaction at room temperature and in the complete absence of moisture. Sodium p-nitrobenzoic and acetyl chloride react vigorously with the evolution of heat, and hence it is necessary to introduce the acid chloride diluted with an inert solvent. To bring about these conditions the apparatus shown in Figure 6 was designed. The reaction unit is fundamentally a Soxhlet extractor modified in such a manner that liquid is transferred from the lower chamber to the upper by means of a vapor lift using nitrogen gas. Procedure A 75 x 150 mm. Soxhlet extraction thimble is soaked over night in reagent grade acetyl chloride, washed with anhydrous ether and dried in the oven at 120° for 15 min. Without being cooled, it is filled with dry sodium p-nitro- FiGURE G > a benzoate and placed in the upper chamber of the modified Soxhlet extractor shown at A. In the flask G was placed 500 ml. of reagent grade chloroform, which was then slowly distilled; the distillate being collected in receiver H until 100 ml. had been col- lected. The stop cock at C was opened and the one at I closed, and 120 ml. of chloroform was collected in the lower chamber of the reaction vessel. Stop cock C was then closed. Reagent grade acetyl chloride in the amount of 150 ml. was placed in flask G and distilled into receiver H until no hydrogen chloride gas was given off through the drying tube J and the reading of the thermometer K indicated that pure acetyl chloride was distilling. (About 20 ml. of the material collected.) Stop cock C was then opened and stop cock I closed. A stream of nitrogen was then. admitted through F in such a manner that a slow steady stream of liquid contained in the lower chamber of the reaction vessel was circulated over the sodium p-nitrobenzoate contained in the extraction thimble in the upper part of the reaction vessel. In the course of 15 minutes, 30 ml. of acetyl chloride was transferred into the reaction chamber. Stop cock C was then closed and the material circulated for an % Before the reaction, the apparatus was thoroughly dried by placing a rubber stopper in the drying tube at B, the stop cock at C closed, and the reaction vessel evacuated through the port at D. Under reduced pressure the appara- tus was flamed and then returned to atmospheric pressure by admission of dry nitrogen through port F. -you additional five minutes. Stop cock L was then opened, and the liquid was transferred to the flask beneath the reac- tion vessel. Seventy-five milliliters of dry chloroform was again GQistilled into the reaction vessel and circulated over the sodium p-nitrobenzoate for 15 minutes. This solution was then added to the main reaction mixture through stop cock L. The solvent was then removed by distillation under vacuum applied through port D. The product was extracted twice with cold carbon tetrachloride, leaving behind a small amount of insoluble residue. An over-all yield of about 30% based upon sodium p-nitrobenzoate was obtained. The purified material melted sharply at 86°C. Shortly after melting, a solid phase separated out of the clear melt. This material was identified as p-nitrobenzoic anhydride and presumably arose through rapid disproportionation of the mixed anhydride. Each of the mixed anhydrides was purified until its spectrum did not change upon three consecutive purification operations. It was noted that distinct and prominent bands of the symmetrical anhydrides and of the acids were always missing from the spectra of the mixed anhydrides. Choice and Purification of Solvent Materials Of the various solvent materials available for use in infrared work, chloroform was found to be the most suitable. -43- For the p-nitro and p-methoxy systems it was a much better solvent than carbon tetrachloride, yet it did not have a strong absorption in the spectral region under investigation. Pure chloroform has the rather distinct disadvantage of undergoing a slow photochemical conversion with the oxygen ‘of the air to form phosgene. The chloroform was purified by taking reagent grade material and washing it several times with 6-8 N sodium hydroxide, then several times with 6 N hydrochloric acid and finally with several portions of water. The solvent was then dried over calcium chloride and fractionated through a 300 ecm. packed column under an atmosphere of nitrogen. The purified chloroform was stored in 25 ml. dark colored glass stoppered bottles at 5°. Only freshly prepared material was used for equilibrium studies, and once a sample was opened to the air it was dis- carded. With the above precautions the conversion of chloro- form to phosgene was not a troublesome factor. Demonstration of the Interconversion of the Symmetric and Mixed Anhydrides When a sample of a mixed anhydride is dissolved in an inert solvent and allowed to stand under anhydrous conditions, its infrared spectrum slowly changes and assumes a new con- figuration. The greatest change occurs in the region of the C-0O-C stretching frequency between eight and nine microns and is characterized by the decrease in size of some of the hy apsorption peaks and the appearance of new peaks rather close to those which decreased. This change in configura- tion of the infrared absorption spectrum for benzoic-acetic mixed anhydride is illustrated in Figure 7. It is possible to identify all of these new peaks with those in the spectrum of one or the other of the symmetrical anhydrides. In a converse manner it is observed that when the two symmetrical anhydrides are mixed in equal proportions, the initial spectrum of the mixture changes in a manner analo- gous to the way that the spectrum of the mixed anhydride changed. The new peaks that arise can be identified with those of the mixed anhydride. If given sufficient time, the final spectrum of the mixed anhydride and the spectrum of a mixture of equal portions of the symmetrical anhydrides be- come identical. Therefore, it is judged that what is being observed is the equilibration of the three anhydride species among one another. 80 50 40 ao OO t=O ene t=O.5 hr t=43hr Figure 7. Infrared Absorption Spectrum in the 8-9 Micron Region t=67 hr 3 =~] i t=92 hr of Benzoic~Acetic Mixed Anhydride as da Function of Time at 25° C. -45- 90 70 60 40 30 %T -U46- The pertinent spectra for the three anhydride systems studied are shown in Figures 8 to 19. In exactly the same way it can be demonstrated that an acid and an anhydride can equilibrate to form a mixture of three anhydrides.and two acids. See Figure 20. Quantitative Measurements Made with the Perkin-Elmer Model 21 Infrared Spectrophotometer The Perkin-Elmer instrument is designed primarily for qualitative use and is most convenient to use in this capacity. When quantitative measurements are sought it is necessary to devise a special operating procedure, which is often quite tedious in execution. When operating in the eight and one half to nine micron region, the resolution control was set so that a slit width of 60-70 microns was obtained. Reliance on the wave length scale was never used in setting the machine at the position of an absorption peak which was under consideration. Rather the outline of the peak was traced out each time, the machine reversed and allowed to retrace its first outline of the peak until the maximum was again reached. At this point of maximum absorp- tion the wave length drivewas stopped and the machine readied for the reading of the optical density. It was found that the position of a maximum might vary about 0.02 microns from day to day. 1% Ov “WdOJOO|YD ul % | | ; oF | aplupAyuy o1;a0y 40 winsj9edS uolWdsosqy paesDsju| Fy | | | °g ounbi4 7 td L | 9 SUOJOIW 1%ZEL ‘apiupAyuy d10ZuEaqg jo. wnsjyoads uoljdsosqy pasdsju| OS F— 7 | ‘6 aunbi4 O9 } ‘ou O08 F— O6 9 SUO491W Ol 6 8 rT] aa w4ojyosojy ul eplapAyuy paxiw 914990-— 910ZU9Q yO WNnsyoedS uUOljdiosSqy pasdAju| "Ol aanbi4 8 Z 9 suoJdIW 1% yl I r “T l l T yl I T | T | O¢€ | | "D9 oGl 40 JINOH | JO} OPtip —-Aysy paxiw 214990 -ds10Zueag BuljoaH Aq paulDjqg a4ny }} -xXlW OD JO winsydadsg uol}dsosqy pasD4juy) Ov fF - {| aunbi4 O09 F— OLE O8 fe) 6 g l | 9 suUdJoIW 1% Ov “UMOJOFO|YD Ul Ploy Bady 40 | winujoadS uoljdiosqy pasdsju| og | | | | “Zl eanbi4 L | | 09 F— 9 Z SUOJOIW 1% TT TT O¢ "WIOJOUOJUD Ul ploy 10Zueg | | JO wnsjoedsS uoljdiosqy paidsjuy | Ob [- | | | ‘e] eunbla Lf : OS F— — SuOJ91W 1% ‘apiupAyuy s1ozuaqkxoujew -d OS | jo wnsjoeds uoljdiosqy pasdsjul “p] ounbi4 09 my) O08 O6 Ol 6 8 Z 9 suoJdIW 1% OS 09 OL 08 O6 OO] Ol ‘apupAyuy “paxil 914900 — 910ZUaqKxouJepw —d yO wnijoadS uoljduosqy pasdsjuy "G] eanBig | 2 9 sUuoJdIW O09 Ol O8 O06 rT Ty tT T T | | 1 | YT TY “wsOjouojyD ul aplpAyuy DIOZUZGOIJIN -~d jo wnsjyoedsS uoljyduosqy pasdsjuy _| “Q| eunbiy 8 Zl 9 sudJoIW 1% ‘wuojosojy) ul apiapkyuy paxiw OG — O1F9IND - DIOZUBQqOIJIN _d jo winsyoodsS uoijdiosqy padidiju| i . | 2| eanbiy l O9 7 OL — O8 O6 Ol 6 Q Z 9 SUOJOIW 1% ANOH | JO} O6GZ 40 aplupAyuYy 21499yY PUD DIOZUSqOsJIN-d Jo s4NyxIW so;oW —-Inbj ud BulyosH Aq paaisaqg a4nyxXIW O JO Wnsjo9ds uoljduosqy pauDdsjul Ov OS F- O09 OL F—- 08 F— 06 | —_ med Ol 6 8 “wJOjosO}UQ ul ] 'Q| aunbi4 l it 9 suosoiw 60 30 40 30 TT microns 3 4 5 }- Figure 19. Infrared Absorption Spectrum of a Mixture [Obtained by Heating Benzoic Acid and Acetic | Anhydride at 75° © in Chloroform. ~ HAc HB, RO i Figure 20 Io Log = Beer's Law Curve for Acetic Anhydride at 8.838 Microns oge- in Chloroform / 06 / O04 -— v4 03 .005/ I Os ee ees eS Qe es | -~50- After the machine was adjusted so that it was reading ve faa ' "Zero point," the absorption of the desired peak, the "Gain,' and "Balance" controls were adjusted with both reference and sample cells in place and filled with solvent (chloro- form). The "sample" light path was closed and the oven carriage was allowed to travel to the low end of the percent trans- mission scale. The "Gain" was then adjusted until a per- ceptible oscillation about an equilibrium point was just obtained. The "Gain" setting producing this effect was be- tween five and six. The pen carriage was then moved on its drive cable until the eguiliorium point of the small oscil- lations coincided with the zero point of the percent trans- mission scale. These adjustments were checked at the con- clusion of each series of measurements. After setting the "Gain" and the “Zero point" controls, the sample light path was opened and the pen allowed TO travel to the high end of the percent transmission scale. The "Balance’ control was then adjusted until a reading of 60-98% transmission was obtained. If the pen carriage is displaced momentarily from its position at the high end of the percent transmission scale by Slowly blocking the sample light path, and then by reopen- ing the light path allowing the carriage to return, it is observed that the pointer does not always return to the same position at the high end of the percent transmission scale -61- that it formerly oecupiec. If a number of observations of this kind are made, it is observed that five or six consecu- tive readings are relatively constant (varying + 0.1 of a sransmission unit) and then that a period of instability is observed when the consecutive readings vary about C.5 4 to 1,0 transmission units. After this brief period of 4 instability, a period of quiescence is again reached, which may or may not be identical with the reading of the former period. Tt was planned that the initial blank reading, tne sample reading, and the final blank reading would all take place during one of these quiescent periods. To try to achieve this the following procedure was used in obtaining the value of the percent transmission of an unknown sample: The sample cell was first filled with chloroform and several readings of @T were ooserved. When three consecu- tive values were obtained which did not differ more than + 0.1 transmission units, the average value was noted and recorded as the first blanx value. The sample cell was removed and quickly filled with the sample. The cell was sabnas reopened. red ch then placed in its holder and tne lignt The pen carriage was allowed to come to rest and then slightly displaced and again allowed to come to rest. Following this, the sample reading was obtained. ‘The sample cell then was removed and guickly flushed and filled with sol- vent and the second blank reading was obtained. If the -62- second blank reading was greater than O.4 transmission units fzom the first, the observations were discarded and the operations repeated. Three sets of these observations were obtained for each quantitative measurement taken, and an average value of these observations was computed. Another variable which must be controlled is the vosi- tion of the cells in the cell holder. The cell holder is slightiy larger than the flange dimensions of the absorp- tion cells. It is necessary that the cells always occupy the same position relative to the light beam for each ob- servation. This was brought about by making certain that the cell flanges always were flush against the side of the cell holder which is nearest the operator. The Determination of the Various Beer's Law Curves To determine the absorption characteristics of the various anhydride species, a given quantity of the anhydride was dissolved in chloroform. Sy dilution of this stock solu- tion and by the use of the procedure which was outlined in the previous section, the optical density of the anhydride at various concentrations was obtained. In general the range of concentrations over which measurements were deter~ mined was chosen so as to cover the limits between which most of the future measurements would be made, A sypical plot of optical density vs. concentration is Shown in figure 20, -03- it is to be noted that while a linear relationship is obtained, the curve does not pass through the origin. There- ?ore, a strict proportional relationship between optical density and concentration does not describe the situation which is presented by several of the anhydride species. It ‘is therefore necessary to use the value of the intercept on the concentration axis to obtain a relationship which is useful over the concentration range of interest. It does not seem possibile at the present time to ascribe a cause for this non-proportionality. It is interesting to note that when another series of measurements were made using cells with a much wider absorption path (1.0 mm.), a linear relationship was obtained which passed through the origin. These cells permitted the use of much more dilute solutions (C.004 M). This suggests that perhaps the relationship is not strictly linear, but due to the insensitiveness of the analytical technique, shis second order effect is not ob- served. All the measurements in this study were made with a Single set of absorption cells. The absorvtion path of the sample cell was 108.5 microns, and that of the blank was 105.3 microns. The data for all the Beer's law curves pertain to the use of these cells and do not strictly apply to any other set of cells. The data obtained from these measurements and a summary of the various Beer's law constants can be found on pages 75 to lil. Gl = Measurements of the Equilibrium Constants The absorption measurements were made as described pre- viously. It was found that at 70°C approximately three days were necessary te insure that the various species present had reached a state of equilibrium, However, it was adopted as standard practice to allow four Gays for the equilibrium state to be established. Measurements at high temperatures were made by sealing the samples under nitrogen in glass ampules mede of 10 mm, pyrex tubing. The ampules were then placed in a small steel bomb (4 war surplus bail out oxygen cylinder). The bomb was then partially filled with chloroform anc inserted in a cottcn seed oil bath, heated by a 200 watt immersion heater and controlled by 4 mercury thermoregulator to + 0.5°C, After the peried of heating, the sample tube was quickly cooled to -10°C and the values of the infrared ab- sorption at the various wave lengths determined as soon as possible. The data from these measurements are presented on pages 112 to 128, Determination of the Rate of Disproportionation The rate of disproportionation was determined by heat- ing a sample of the mixed anhydride at 100°C and withdraw- ing samples of the hot material at various times for measure- ment of the mixed anhydride content. Since the mixed anhydrides react rather rapidly with moisture, it is neces- sary to carry out the heating and sampling under anhydrous -65- conditions. To bring this about the special apparatus shown in Figure 21 was constructed. The chamber where the samples of mixed anhydride were heated while undergoing disproportionation is shown at A. The temperature is controlled by the flow of steam through B-C, and recorded by thermometer D. A sample is withdrawn for measurement by applying a slight positive pressure of nitrogen at E. Stop cocks F and G are adjusted such that the anhydride can flow out of exit port H. The mixed anhy- dride is allowed just beyond the stop cock G irf H and then F is turned 90°. G is turned 180°. When the desired time has arrived to take a sample, stop cock F is turned another 90° and the mixed anhydride allowed to flow directly into a volumetric flask containing chloroform. The exit tip J is placed under the surface of the chloroform, so that the transfer of the mixed anhydride is made without it ever coming in contact with the air. After the transfer is made, the sample is quickly adjusted to volume and then quenched to -10°C. The anhydride sample, when passing from the reaction zone to the measuring tube between stop cocks F and G, must pass outside of the steam heated zone. To avoid errors introduced by this passage, the external section is wrapped with a nichrome heating element and a thermocouple attached to the glass wall at the center of the external passage. The whole array is wrapped with ashestos tape. The temperature in the external segment is controlled by varying the passage of current through the heating element in response to the FigURE a1 (-) D & —_o \\ hie ¢ | | ~T ola LA] ar, -67- thermocouple resding. The volume of the tube between stop cocks F anc G was found te be 0.1154 em, The semples were kept in the -10°C bath until immedi- ately before they were placed in the sample cell of the infrared spectrophcetometer, | The data obtained by this procedure are shown on pages 129 to 138. | Determination of the Nature of the Reaction of the Mixed Anhydride with Nucleophilic Agents In order to determine the composition of the product mixture obtained from the reaction of a mixed anhydride with p-toluidine, recourse was again made to the infrared spectrophotometric analytical technique outlined in 4 pre- vious section. With p-methoxybenzoic anc p-nitrobenzoic- acetic mixed anhydrides, the absorption bancs associated with the characteristic vibrations of the methoxyl anc nitro group served as a convenient means for measuring the amount cf N-(p-methoxybenzoy1)-p-toluidine enc of N-(p-nitrobenzoyl)- p-toluidine. N-Benzoyl~p-toluidine was determined by the use of its carbonyl stretching absorption. The frequency of this absorption corresponded to the same absorption of the acetyl compound, but its intensity was greater, The acetyl compound was determined by the use of a relatively minor peak in its infrared spectrum. The assignment of this peak to a specific interaction does not seem possible at this time. The generalized procedure for determining the acetyla- tion ratio is as follows: A 0.05 formal solution of the mixed anhydride in the. organic solvent was prepared, and an equal volume of an O.11 molar solution of p-toluidine in the same or different solvent was added. The homogeneous mixture was allowed to stand for 24 hours and then was evaporated to dryness. The solids were then treated with 10% excess of the amount of 1 N sodium carbonate necessary to convert all the free actd present to its sodium salt. The mixture evaporated to dryness at room temperature. The mixture of the sodium salts, p-toluidine, and the toluidides was then extracted with two 50 ml. portions of hot benzene. The benzene solu- tions were evaporated to dryness and the dry solids treated with a sufficient quantity of 6 N hydrochloric acid to neutralize all of the p-toluidine present. The heterogeneous mixture was again evaporated to dryness at room temperature. The mixture of p-toluidine hydrochloride and the toluidides waS again extracted with two portions of warm benzene. The benzene was again removed by evaporation. Finally the re- maining solics were taken up in chloroform, and from this solution the value of the acetylation ratio was obtained. This procedure removes any chance for the alteration of the acetylation ratio, which might occur through several par- titions between an aqueous and organic solvent. The results of this procedure are shown on pages 149 to 154. -69- The Determination of the Rate of Hydrolysis of the Mixed Anhydrides The procedure for this determination has been well > 1 worked out 62218) It consisted, as modified for the present situation, of dissolving the mixed anhydride in a tetra- hydrofuran-water mixture and pipetting samples of this solu- tion at given times into an arresting solution, The arrest- ing solution was composed of aniline and tetrahydrofuran, Its composition was adjusted so that a homogeneous phase was maintained upon the addition of the anhydride solution. The concentration of aniline in the arresting solution was at least twice that of the anhydride. The aniline reacted very quickly with the anhydride present in the aqueous sol- vent. Titration of the arresting solution with standard hydrochloric acid easily gave the concentration of the anhydride in the aliquot portion. The values of the apparent first order rate constants listed on page 155 have been corrected for the amount of tetrohydrofuran used to obtain homogeneity in the initial solution, The Estimation of the Probable Error in the Determination of an Equilibrium Constant The probable error in the slope and intercept of a line drawn through a seriss of points, which deviate from the line in a random fashion, can be estimated by the (19) following series of formulae. -~70- For y = + bX fOr y a an . ay 19) Probable errors in 2a and b are R= Ve | Exe / Py= Ae VW hong | ne=0.6TH5 [Eat Da m@xXj -(Z%)* (1-2) vs Using the above formulation the probable errors in the slopes and intercepts of the Beer's law functions were computed for the benzoic-acetic anhydride system and are compiled in the table: Table 9 Probable Error in the Beer's Law Functions for the Benzoic-Acetic Anhydride System Wavelength 8.224 microns 8.888 microns 8.690 microns Substance Slope Intercept Slope Intercept Slope’ Intercept Acetic 0.360 0,007 3.49 0,024 ~ 0.662 0.009 Anhydride £0,002 +0, 004 +0.05 +0.002 +0.,017 40.005 Benzoic 3.598 0.000 0.151 0.000 0.0912 0,000 Anhydride +0,051 +0.006 +0,.00140,.002 +0, 000440. 0007 Mixed 0.290 0.004 0.663 0.004 - 2,292 0.005 Anhydride +0,006 40,002 +0, 004+0.003 +0,05. +0,.007 For the final computation of the concentration of an anhydride species the following third order determirant must be solved: Oo Whearo P. Fan n [omyt za, + [awkgda] b+ Lowhgel a] C= Ne [omlylr] ag + Lombylar] b, + [amide] Ca= Na [ovbydagst Lamp] by + Leeda] = Ns Wher Ny, Nz, ame Ns aro-the erirectid optical dimailien The probable error in a function (f) derived from a f \ P eae . . . 20 series of quantities (S;) whose error is Q; is given py $V! PRE, mfr Ie et) Therefore, for the anhydride species Anhyd I the prob- able error would be given by: 2. ) 4, Cc, a, 5, C, No by co{- PRE #42 b, Ce aq + 3 bs Cs %5 by Cs Qa, 5, eC; Ae b, ¢, | 2s b3 Cs N, b, ¢, Na b, ¢, | = 2. CCNY ple) ] + (om) (aye) [(caNcb, Cand] - - N3 b3 Cy ~--- 4 [ON ( b,\(4e) J+ [Onley db 3] 4+ LG) (b,\(dul) /* ~72- when the acrove calculations were carried out ror the data on the tcp half of page 120, the following results were obtained: (Acetic Anhydride) = 0.CB26 + 0.0015 (Benzoic Anhydride) = 0.0340 + 0.0015 (Benzoic-Acetic Mixed Anhydride = 0.0466 + 0, 0041 Equilibrium Constant = 4.31, + 0,144 e ay FP QO tr & L. QQ -73- REFERENCES Behal - Ann. Chem. Phys. 19 274 (1900) Kilpatrick - J.A.C.S, 52 1418 (1930) Michael - J. Pract. Chem. 60 364 (1899) S$. Breslow, C. R. Hauser - J.A.C.S. 61 786 (1935 GC. Fuson - "Advanced Organic Chemistry" - page 197 (1950) John Wiley & Sons, N.Y. . Wieland, R. Sehring - Ann. der Chemie 569 117 (1950) R. Vaughn, R. Osato - J.A.C.S. 74 678 (1952) Brown, I. Trotter - J. Chem. Soc. 87 (1951) Calingaert - J.A.C.S. 61 2748 (1939) Emery, V. Gald - J. Chem. Soc. 1447 (1°50) M. Pedder, J. C. Lattlow, F. Bourne - J. Chem. Soc. 2006 (1954) | P. Hammett - Physical Organic Chemistry cage TT (1940) MeGraw Hill Book Co., N. ¥. | We Wheland - The Theory of Resonance page 73 (1944) John Wiley & Sons, N.Y. L. Franklin - J.A.C.S. 72 4279 (1950) . A. Evans, J. Huston, T. Norris - J.A.C.S. 74 4985 (1954) M. Buswell, W. H. Rodebush, M. F. Roy - J.A.C.S. 76 2239 (1954) Berliner, L. H. Altashul - J.A.C.S. 74 4110 (1952) S. Hurd, B. K. Dull - J.A.C.S. 54 3427 (1932) -74- + K. Verkade - Red. Trav. Chim. 35 399 (1915) Margenau ~ Murphy "The Mathematics of Physics and Chemistry" page 502 (1943) Macmillan and Co. R. Livingston "Physico Chemical Experiments” page 22 (1948) Macmillan Co., N.Y. -75- Summary of Data for tne Benzoic-acetic Anhydride And Benzoic-acetic Acid Systems Principal Wave lensth Benzoic-acetic mixed anhydride ---- 8.690 microns Acetic anhydride ------------------ 8.888 microns Benzoic anhydride ----------------- 8,224 microns Acetic acid ----------------------- 5.823 microns Benzoic acid ---------------------- 7.570 microns Beer's Law Curves A. Benzoic-acetic mixed anhydride 1. At mixed anhydride wave length (8.690 microns) Log 42 = 2.292(M-B,) + 0.0050 At acetic anhydride wave length (8.888 microns) Log 42 = 0.6625(M-Bz) + 0.0100 At benzoic anhydride wave length (8.224 microns) Log 42 = 0.2901(M-Bz) + 0.0040 At acetic acid wave length (5.823 microns) Log =o = 0,2818(M-Bz) + 0.0000 At benzoic acid wave length (7.570 microns) Log #2 = 0.2550(M-Bz) + 0.0000 Benzoiec anhydride i. At mixed anhydride wave length (8.690 microns) Log 42 = 0,0912(Bz,0) + 0.000 a At acetic anhydride wave length (8.888 microns) Log #2 = 0.1507(Bz,0) + 0.000 2 1 -~75- At benzoic anhydride wave length (8.224 miczons) { t+ 0 Log = 3.5¢08(Bz,0) + 0,000 At acetic acid wave length (5.823 microns ) Log #2 = 0.3090(Bz,0) + 0.000 At benzoic acid wave length (7.570 microns ) Log £2 = 0,2260(8z,0) + ¢.000 Acetic Anhydride 1. 2. Os . A, on) At mixed anhydride wave length (8.65¢0 microns) Log 22 = 0.6623(Ac,0) + 0.0089 As benzoic anhydride wave length (8.224 microns ) Log ae = 0,3602(Ac,,0) + 0.0075 At acetic anhydride wave length (8.888 microns) Log #9 = 3.491(Ac,0) + 0.024 At acetic acid wave length (5.823 microns) Log a 0.0575(Ae,0) + 0.000 At benzoic acid wave length (7.570 microns) Log io = 0.0468(Ac,0) + 0.000 D. te -(7- Acetic Acid 1. 2. UW ° At mixed anhydride wave length (8.690 microns) Log #2 = 0.0191(HAc) + 0.C050 At acetic anhydride wave length (8.888 microns) Log 22 = 0,0339(HAc) + 0.0030 At benzoic anhydride wave length (8,224 microns) Log 2 = 0.0384 (HAc) + 0.000 | At acetic acid wave length (5.823 microns) Log a2 = 2,140(HAc) + 0.000 At benzoic acid wave length (7.570 microns) Log an = 0.0778(HAc) + ©.0065 Benzoic Acid 1. Po a At mixed anhydride wave length (8.6¢°0 microns) Log i = 9,0530(HBz) + 0.900 At acetic anhydride wave length (8.888 microns) Log #2 = 0.2458(HBz) + 0.000 At benzoic anhydride wave length (8.224 microns) Log 2 = 6, 0404 (HBz) + 0.COO At acetic acid wave length (5.823 microns) Log a = 1.710(HBz) + 0.0209 At benzoic acid wave length (7.570 microns) Log =e = 0.7790(HBz) + 0.000 -78- Simultaneous Equations Derived from the Beer's Law Data A. No Acid Present 1. 1. rh Wy At 8.224 microns 3.598(Bz50) + 0.3602(Aco0) + 0.2901(M-Bz) = g Log 18 ( To - 0.0115 At 8.888 microns Hl 0.1507 (Bz,0) + 3.491(Ac,0) + 0.6625 (M-Bz ): Log #2 - 0,034 At 8.690 microns i] 0.0912(Bz50) + 0.6025(Aco0) + 2.292(M-Bz ) Log ae - 0.014 Acids Present At 8.690 microns 2,292(M-Bz) + 0,0912(Bz50) + 0.6025(Ae50) + O.0191(HAc) + 0.0530(HBz) = Log fe ~ 0,019. At 8.888 microns 0.6625(M-Bz) + 0.1507(Bzo0) + 3.491(Aco0) + 0.0339(HAc) + 0.2458(HBz) = Log ae - 0.0370 At 8.224 microns | Q.2901(M-Bz) + 3.598(Bzo0) + 0,3602(Acp0) + 0,.0384(HAc) + 0.0404(HBz) = Log 2 --O0.0115 At 5.823 microns 0.2818(M-Bz) + 0.3090(Bz50) + 0.3602 (Aco0) a 2,140(HAc) + 1.710(HBz) = Log #2 - 0.0200 At 7.570 microns | 0.2550(M-Bz) + 0.2260(Bz20) + 0.0468(Ac50) + 0.0778(HAc) + 0.7790(HBz) = Log to - 0,0065 -~79G- Beer's Law Curve For Acetic Anhydride At 8.888 Miecrons Concentration moles per liter Blank Sample Blank %T 92,6 16.1 92.8 17.367 0.2122 02.6 15.9 92.4 17.189 G2.4 15.9 $2.2 17.226 O7.0 23.6 O7.0 4. 30¢ 0.1700 87.9 23.6 S7.2 24,317 66.9 23.6 G7.1 24.329 90.6 23.9 GO.8 26.350 O.1591 90.9 e4.0 91.3 26.344 O1.7 P41 91.5 26.310 03.6 37.4 93.7 39.935 0.2061 93.5 37.5 S4.0 40.900 S3.5 37.4 S40 39.808 O14 51.1 62.2 55.969 ©.0636 Oo1.0 451.1 91.1 56.123 91.2 51.1 91.2 56.030 C1.5 61.7 91.4 67.468 0.0424 S1.2 61.6 91.3 67.506 G1.2 61.7 91.3 67.616 91.6 74.5 61.6 81.331 0.0212 Ol.1 7ALL 01.0 81.383 91.2 74.0 S1.0 81,229 Log 42 = 3.491(Ae50) + 0,024 Ave. 40 Loge ave. 5.7934 41109 3.8021 0.580021 0.170202 9.089838 -80- Beerts Law Curve For Acetic Anhydride At 8.22 Microns In Chloroform Concentration moles per liter Blank Sample Blank &%T Ave ,12 Log ave +0 ; 02.7 55.0 93.0 59.235 . 0.6166 93.3 55.2 93.0 59.259 1.6850 0.22660 93.0 55.1 93.0 59.247 oo 93.0 64.9 93.0 69.784 Oo. 4144 93.1 65.0 93.4 70.270 1.4304 0.155460 93.2 64.9 93.1 69,672 92.7 70.8 92.5 76.457 0.3083 02.6 70.8 92.7 76.416 1.3099 0.117237 02.2 70.2 92.2 76.138 93.0 77.0 93.2 82.706 ; 0,2122 93.1 77.4 93.4 83.002 1.2066 0, 081566 93.3 77.3 93.4 82.806 90.7 82.3 91.0 90.588 0.1061 91:0 82.3 91.1 90.389 1.1044 0.043126 61.0 83.3 91.9 90.542 Log 42 = 0.3602(Ac,0) + 0.0075 Beer's Law Curve For Acetic Anhydride At 8.600 Microns In Chloroform Concentration , . to moles per liter Blank Sample Blank ol Ave, 48 Log ave = 91.8 48.7 92.0 52,992 ; O.4e44 91.7 48.6 91.8 52.970 1.8882 0.276046 91.7 48.5 91.6 52.918 91.3 55.0 90.7 60.439 0.3183 90.8 54.5 91.2 59.890 1.6615 0.220400 01.3 54.9 91.0 60.230 92.0 65.7 92.3 71.296 0.2122 92.0 65.0 91.4 70.883 1.4069 0.148260 01.3 64.8 S1.1 71.052 93.9 78.3 93.5 83.564 0.1061 93.0 78.0 93.0 88.870 1.1937 6.076892 92.4 77.5 92.4 83.874 oo.4 80.7 GO.7 89,122 0.06375 00.2 80.3 90.1 89.073 1.1222 0.050068 91.0 81.1 91.0 89,120 Log #2 = 0.6623(Acs0) + 0.0089 -~B1- Beer's Law Curve For Acetic Anhydride At 5,823 Microns In Chloroform Concentration r To moles per liter Blank Sample Blank %T Ave. =p Log ave.7 . 90.2 71.6 90.5 79.247 1.6958 90.3 71.5 89.9 79.356 1.2609 0.100685 90.0 71.3 89.8 79.310 eee 90.5. 76.5 90.3 84,623 | 1.2718 90.3 76.2 90.3 84.385 1.1183 0.073065 o0.4 76.4 90.5 84.46 91.3 79.3 91.2 86.904 1.0599 91.4 79.3 91.2 86.856 1.1510 0.061080 91.8 79.8 91.9 86.880 90.2 80.5 90.5 89.196 0.8479 90.0 80.2 90.2 89.101 1.1216 0.049638 90.1 80.3 90.0 89.172 Log 2 = 0,0575(Ac,0) + 0.000 Beer's Law Curve For Acetic Anhydride At 7.570 Microns “n Chloroform Concentration To “moles per liter Blank Sample Blank %T Ave.i9 Log ave = CO Ly 93.5 78.0 93.5 83.422 oe | 1.6958 92.6 78.1 93.5 83.484 1.1982 0.078532 93.3 78.0 93.4 83.556 94.8 82.7 S4.6 87.382 1.2718 o4.7 82.8 94.5 87.480 1.1447 0.058796 03.7 81.9 o4.0 87.266 95.9 85.5 95.7 89.248 1.0599 94.3 94.3 94.6 89.253 1.1208 0.049508 95.0 84.7 94.8 89.251 95.0 86.6 95.0 91.157 — 0.8479 95.0 86.8 $5.3 91.224 1.0949 0.039371 95.2 86.9 95.2 91.281 Log +e = 0.0468(Ac,0) + 0.0000 -Oc¢c- Seer's Law Curve For Benzoic Anhydride At 8.224 Microns In Chloroform Coneentration +5 moles ver liter Blank Sample Blank &%T Ave = T 97.5 35.6 97.5 36.512 O.1210 o7.44 35.6 97.6 36.512 2.7385 563.8 38.8 93.8 41.364 0.1061 ch. 36.0 G4.2 AL.4as5 24124 93.5 39.0 93.7 41.655 97.5 43.3 97.5 44.410 0.0275 o7.4 43.3 97.6 Hk.410 2.2513 $8.0 43.5 97.8 44.433 93.2 46.5 93.1 49.919 9, 08488 S3.C 46.2 93.1 46,650 2.0097 02.9 46.2 93.0 45.704 92.4 54.8 92.8 59.17¢ 0.06366 92.0 54.5 62.5 56.078 1.6922 92.6 54.6 62.4 59.027 92.8 65.0 92.7 79.080 0. O4240 02.7 64.9 93.2 65,822 1.4307 93.0 64.5 93.0 69.784 92.0 89.1 92.5 ©£6.585 0.00408 92.3 89.5 92.4 66.913 1.033¢ o2.0 89.0 92.2 6.663 To Log 7 = 3.598 (Bz50) + 0,000 Log ave. 0.352437 0. 303034 0.228530 oO Ke wi yn Noa a? ©O 0.014478 5 ° ~83- Beer's Law Curve For Benzgoic Anhydride At 8.388 Microns In Chloroform Concentration moles per liter Blank Samole Blank er ave. 42 hog ave +2 65.5 85.4 95.7 89.330 0.3183 65.7 85.3 95.6 80.179 1.1207 0.04993 oh5 84.4 Gh.8 80.170 92.5 86.1 92.9 93.333 9.19998 61.8 86.0 $1.7 63.732 1.0691 9.029020 92.0 86.0 $1.9 93.526 92.5 87.5 $2.5 4.554 0.15612 02.7 87.5 S2.5 S4.402 1.0576 0.024322 92.6 87.5 92.4 S4.5¢4 93.5 90.2 94.0 $6,223 0.10610 clh.c 90.4 S4.0 G6.170 1.0392 _ 0.016702 G4ULO 80.5 94.0 S6.276 92.2 89.5 92.3 $7.018 0.0795 Q2.1 89.5 92.2 97.124 0297 0.011864 92.2 8.6 92.2 97,180 Log 7 = 0.1507(Bz5,0) + 0,00CO Beer's Law Curve For Benzoic Anhydride At 8.690 Microns In Chloroform Concentration Lo moles per liter Blank Sample Blank #7 Ave. 48 Log ave .>- 02.6 90.7 92.8 97.842 0.1061 02.5 60.5 92.7 S7.732 1.0222 0.009536 92.5 99.6 92.6 97.893 62.0 ¢cO.4 92,2 98.154 0. 09544¢ C2,1 SO.4 92.0 98.100 1.0197 9.008471 92.2 $0.3 S2.2 97.939 92.5 91.0 92.8 $8,215 0.08488 92.6 G1.0 92.5 68. 325 11,9173 o.oc744e 02.5 S1l.1 92.8 98.327 Log £2 = 0,0¢12(Bz) + 0.000 -Bh- Beer's Law Curve For Benzoic Anhydride At 5.823 Microns In Chloroform Concentration moles per liter Blank Sample Blank oT Ave te Log ave. 40 96.0 57.4 96.4 59.667 0.7250 95.8 57.3 96.0 59.749 1.6752 0.22h062 95.9. 57.3 96.2 59.656 95.5 70.0 96.0 73.107 0.4350 95.5 69.5 95.5 72.774 1.3673 0.135866 94.5 69.4 94.3 73.516 94.6 73.0 94.8 77.085 0.3625 O4.9 73.1. 95.1 76.906 1.2982 0.113340 O4.7 73.0 94.7 77.085 96.5 77.9 96.5 80.725 0.2900 95.5 78.0 95.7 81.589 1.2309 0.090225 95.4 77.9 96.0 81.400 . 95.0 84.0 96.0 87.958 0.18125 95.5 84.0 95.4 88.004 1.1362 0.055446 95.5 84.1 95.5 88.062 i fe) og | { : ae = 0.3090(Bz50) + 0.000 ~85- Beer's Law Curve For Benzoic Anhydride At 7.570 Microns In Chloroform Concentration moles per liter Blank Sample Blank 0.7250 95.1 64.6 95.1 95,0 64.7 95.0 O4.4 °° 75.1 94.2 0.4350 S4.1 75.1 4.3 O44 75.0 94.2 93.7 77.4 93.7 0.3625 93.9 77.5 94.0 O4u.O 77.4 93.7 93.8 80.5 94.0 0.2900 93.9 80.6 94.0 94.2 80.4 94.4 93.5 85.2 93.7 0.18125 93.4 85.2 93.7 93.7 85.2 93.5 93.7 86.9 93.9 0.1450 93.6 87.0 94.0 O41 86.9 93.5 Log £2 = 0,2261(Bz,,0) al 67.962 67.928 68.105 79.639 79.723 79.533 82.604 82.490 82 472 85.729 85.790 85.259 91,025 91.074 91.025 92.643 92.750 92.643 + 0,000 Ave, Lo ay 1. io e LT OT he) .e} Ut —~ -cll7y . 1683 0674 Log Ave aa To . 167523 ~098885 . 083392 . 067551 O40760 . 032380 -86- Beer's Law Curve For Benzoic-acetic Mixed Anhydride at 8.6°0°C Microns Ta Chioroforn Concentration moles per liter Blank Sample Blank %7 Ave. 38 Log ave 40 98.6 21.3 98.7 21.591 ; 0.29065 08.0 21.2 98.0 21,632 4,6264 0. 665246 68.0 21.2 S8.1 21.621 G1.3. 21.8 S1.4 23.864 . - 0, 2645 61.3 21.6 01.3 23.877 4.1867 0.621870 G1l.2 21.8 Sl.1 23.916 ; 08.4 25.6 ¢8.3 26,029 0.23276 08.2 75.5 GB.1l 25.680 3.8454 0. 5840u4 G8.0 25.5 C8.1 26.007 O1.5 31.4 00.6 34.666 0.1980 66.8 32.5 $0.5 34.7490 2.8841 0.460015 90.1 32.2 90.4 34.570 92.3 45.3. 92.4 4o,05e2 O.13225 $2.0 45.1 62.2 48.968 2.0391 9 0.309441 61.8 45.0 S1.¢ 48.992 Sl.2 53.5 61.6 58.46¢ O,095S0 Ql, 3.4 °0°¢6G1.4 58.520 1.7063 9,232818 O1.e 53.4 c1.3 58.520 O1.1 62.0 91.4 67.945 0,0661 91.3 62.0 91.2 67.°45 1.4723 0.168003 O1.4 62.0 61.3 67.870 60.5 77.0 91.0 84,848 0.02655 91.5 76.8 91.60 84.164 1.1817 O.C7254S 90.7 77.0 90.8 84,848 Lo i} NO MO mM ~~ Lerncl se | O oe) 1o3) Log -S7- Beer's Law Curve For Benzoic-acetic Mixed Anhydride At 3.224 Microns In Chloroform Concentration x | To moles per liter Blank Sample Blank eT Ave. == hog ave.F 91.5 60.9 91.5 66.557 0.5745 91.4 60.9 91.5 66.592 1.5013 0.176467 91.5 61.0 91.5 66,666 ; ; 91.2 67.7 61.5 74.110 0.4220 91.5 67.7 91.5 73.989 1.3505 0.130495 $1.5 67.7 91.4 74.029 91.5 69.5 91.5 75.956 . 0.3591 O4.1 71.4 93.9 75.957 1.3164 0.119392 91.4 69.4 91.5 75.888 92.2 75.6 92.0 82.084 0.2680 91.5 75.5 92.0 82.288 1.2163 0.085038 . 92.0 75.5 92.0 82.065 oo 93.5 84.5 93.1 90.568 0.1325 92.6 83.8 92.7 90.447 1.1063 0.043877 92.7 83.8 92.6 90.447 Log x2 = 0,2901(M) + 0.0040 Beer's Law Curve For Benzoic-acetic Mixed . Anhydride At 5,605 Microns In Chloroform Concentration moles per liter Blank Sample to am o ) at PSX Hl Ave. 29 Log ave.== i, 93.5 43.5 93.5 46 52h 0.5745 — 93.5 43.6 93.6 46.606 2.1476 0.331950 93.6 43.6 93.7 46.556 o4.2 54.0 94.3 57.355 0.4220 94.0 54.0 94.2 57.285 1.7444 0.24165 O4.5 54.1 94.2 57.339 . 93.6 64.2 93.9 68.480 0.2680 93.0 64.3 93.2 69.065 1.4553 0.16295 93.0 63.7 93.0 68,404 93.3 77.1 93.6 82.504 0.1325 93.9 77.8 94.0 82.810 1.2136 0.084076 93.5 77.2 $3.7 82.478 93.5 84.6 93.8 90.336 0.06625 93.8 84.5 SO4.1 89.c41 1.1102 0.045398 93.9 84.5 Sh.o 80.S41 Log #2 = 0,6625(M) + 0.0100 ~88- Beer's Law Curve For Benzoic-acetic Mixed Anhydride At 5,023 Microns In Chloroform Concentration moles per liter Blank Sample Blank 4T Ave 20 Log ave 42 98.5 59.3 98.5 60,203 0.7835 98.6 59.2 98.6 60.040 1.6637 0.221072 98.5 59.3 08.6 60.172 97.0 63.6 97.0 65.567 0.66125 96.9 63.7 97.2 65.636 1.5246 0.183154 97.2 63.7 97.1 65.568 95.3 68.5 95.6 71.765 0.5290 95.4 68.5 95.5 71.765 1.3926 o,14yune 95.5 68.4 95.6 71.585 98.0 74.5 98.3 75.904 0.39675 O7.9 74.5 98.0 76.059 1.3084 — 0.116742 07.8 74.6 97.7 76.317 95.5 81.5 95.4 85.385 0.2645 95.4 81.5 95.2 85.519 1.1724 0.069078 95.2 80.9 95.2 84.978 Log #2 = 0,2818(M) + 0.000 Beer's Law Curve For Benzoic-acetic Mixed Anhydride At 7.570 Microns In Chloroform | — Concentration . - moles per liter Blank Sample Blank 4%T Ave 40 Log ave.40 97.1 65.0 97.2 66.906 | 0.66125 97.0 65.0 97.3 66.906 1.4044 oO.174466 97.3 65.1 97.2 66.940 96.5 70.0 96.7 72.463 0.5290 7.0 70.0 97.0 72.164 1.3819 0.140378 96.5 70.0 96.7 72.463 97.0 82.1 97.3 84.508 0.2645 97.2 82.1 97.1 84.508 1.1846 0.073572 97.4 82.0 97.3 84,232 97.1 86.6 97.2 89.140 0.19838 97.2 86.6 97.2 89.094 1,1227 0.050263 o7.4 86.7 97.5 88.968 97.6 CO.7 96.4 93.989 0.13225 96.3 90.1 96.2 93.610 1.0701 0.029421 96.0 89.9 95.8 93.743 Log 42 = 0,2550(M) + 0.000 Beer's Law Curve For Acetic Acid At 8.6900 Microns In Chloroform Concentration + To moles per liter Blank Sample Blank $T Ave #2 Log ave == 98.2 85.4 08.1 87.009 1,424 98.2 85.4 97.9 87.0908 1.1491 0.060358 98.0 85.5 98.3 87.2111 | 97.5 87.6 97.3 89.938 1.187 o7.3 87.5 97.2 86.974 1.1115 6.045605 67.7 88.0 97.9 8¢6.97¢ 07.0 89.7 66.8 92,569 0.7120 96.7 89.9 66.9 62.871 1.0783 0.03274 S6.1 89.1 96.0 ¢e.764 Log = = 0.0191(HAc) + 0.005 Beer's Law Curve For Acetic Acid At 9,086 Microns In Chloroform Concentration To moles per liter Blank Sample Blank @T Ave. 19 Log ave.>> 98.5 78.4 98.4 ©, 634 1.424 C8.4 78.4 68.2 79.755 1.2528 0.997880 98.0 78.5 98.1 80,061 08.6 3.1 98.3 84,408 1,187 908.3 82.9 98.0 84.462 3.1844 0.073408 58.0 82.8 98.2 84,403 95.5 85.1 95.2 89.250 O,.7120 G5.5 85.2 95.6 869.167 1.1213 0o.oho72h 95.5 5.2 95.6 86.167 0.3560 95.5 86.4 95.7 93.514 1.0678 0.028488 95.6 89.5 95.2 93.913 0.0339(HAc} + 0.003 eI Beer's Law Curve For Acetic Acid At 8.224 Microns In Chloroform Concentration moles ver liter Blank GHL6 1, 4ed GLO $3.9 G3 AL 0.1187 $3.9 G40 $3.6 0.3560 O3.7 63.3 Log =- = 72.9 Gh e 73.1 94.2 73.0 G3.7 Oo N ON ‘@) FW * Wm) OY 1ee) (ai LOO 88.3 $3.9 88.2 93.6 88.4 $3.9 C.0384(HAc) o4, 186 94.080 S4..1.92 To Log ave 0.11025 6.054078 0.026162 Beer's Law Curve For Acetic Acid At 5.823 Microns In Chloroform Concentration To moles per liter Blank Sample Blank 47 Ave, 29 Log ave. 94.5 19.4 S4.5 20,526 0.3560 94,0 19.3 S48.C 20.531 4.8732 6.687818 S41 16.2 94.2 2a.4ac . 95.4 23.7 95.2 24.868 0.2848 95.C 23.6 95,1 24,826 4.0268 0.504058 o1.8 23.5 94.7 2h,802 92.0 25.9 91.5 28.383 0.2541 91.8 25.9 $1.9 28.198 3.5360 0.54851 91.7 25.9 91.6 28.25¢ 93.4 31.6 $3.0 23.905 0.2136 G3.0 31.5 93.2 33.824 2.9509 c.46o955 92.2 31.6 93.1 33.923 96.2 38.3 96.5 39.750 0.1825 06.5 38.4 96.8 36.730 2.5157 ©.406590 96.0 38.2 96.1 39.770 93.C 37.5 92.6 40 .4os 0.1762 92.8 37.5 92.5 4o.474 e2.47ke 6.393436 93.1 37.6 93.2 40.365 | 95.2 45.1 95.6 47.274 . 0.1460 95.7 45.2 95.5 47.280 2.1159 6.325499 G4.8 44.9 65.0 47.312 93.2 48.8 93.1 52.388 0.1250 93.7 46.0 93.5 52.322 1.9100 0.281030 03.5 48.9 93.3 52.355 93.5 63.9 93.5 67.375 . 0.0701 93.3 62.7 92.9 67,346 1.4835 O.1712¢0 02.5 62.7 92.9 67.491 92.5 75.9 62.5 82.054 0.0350 602.2 75.5 92.0 81.976 1.2202 0.086432 02.60 75.4 92.3 81.823 Log #2 = 2,140(HAc) + 0.000 (Concentration Limit 0.24 molar) -92- Beer's Law Curve For Acetic Acid At 7.570 Microns In Chloroform Concentration noles per liter Blank Sample Blank er Ave 50 Log ave to 95.0 70.0 95.4 73.529 1.5150 95.6 7C.1 95.5 73.364 1.3642 0.134874 95.8 69.9 5.7 73.002 93.0 74.1 93.4 79.506 1.1120 96.2 76.8 66.5 79.709 1.2579 0.109646 96.5 76.5 96.5 79.274 C44 80.7 94.5 85.442 0.727 93.4 80.2 93.4 85.867 1.1644 0.066102 93.4 79.9 93.6 85,454 95.9 85.0 95.9 88,633 0.539 06.0 85.1 96.3 88.507 1.1281 - 0.052348 05.8 85.0 95.7 88.772 96.5 88.7 96.8 91.774 0.359 66.2 88.5 96.1 92,043 1.0871 0.036270 96.0 88.4 95.9 92,131 Log #2 = 0.0778(HAc) + 0.0065 Beer's Law Curve For Benzoic Acid At 8.660 Microns In Chloroforn Concentration ro moles per liter Blank Sample Blank {%T Ave, 20 Log ave. 65.2 86.8 94.8 61.368 0.7266 95.0 86.9 65.1 91.425 1.0956 ©.039650 95.3 86.7 95.0 94.023 $6.8 S0.5 O7.1 93.347 0.58928 CT. 90.7 S7.3 93.321 1.0715 0.C30190 S7.2 90.8 S74 93.319 07.5 93.8 $7.5 96.205 0.3183 67.8 93.9 S7.7 96.061 1.0391 0.01661 S7.0 93.7 $7.3 96.448 Lo ‘ i\ et Tt ‘ e Ve Log 7 = ©.0530(HBz) + 9.0C0 Beer's Law Curve For Benzoic Acid At 8.888 Microns In Chloroform Concentration ; r To moles per liter Blank Sample Blank gat Ave. = Log ave > c7.2 64.8 66.9 66.769 0.7366 o7.0 64.9 S7.1 65.872 1.4966 0.275004 o6.9 64.7 06.8 66.604 06.2 69.5 96.3 76.207 0.58928 96.5 69.6 96.2 76.236 1.3344 0.741268 66.0 69.4 $6.1 72.254 96.2 75.1 $6.5 77.944 0.43196 $6.5 75.5 96.1 78.400 1.2751) 1.906004 96.0 75.2 96.3 78.211 396.0 81.2 S6.0 84.687 0.3183 96.5 81.1 $6.3 84.128 1.1852 0.073792 66.3 81.4 96.8 84,308 - Lo cylier OO las Log == = 0,2458(HBz) + 0.000 -Che Oat Beer's Law Curve For Benzoic Anhydride At 8,234 Microns In Chloroform Concentration moles per liter Blank Sample Blank %T - O77. 1 0.7366 O75 O7.5 ran Sard ©) s WI 8) KD m Oo IM CN QI © Co Oo Loe) e ee) CA . Log == LOD OG © e CAN ON LOL KE Nh MA eat S1.7 g2.0 S1L.S OD t Oo? dee / ji 7 LONOKE Onn KO SOW UI OVW 4 63.161 62 83.168 230 «§3.018 20 S4,633 25 Ch 601 6 Ch 64h 97.023 97.035 O7 .032 LO GI CO = 0,0404(HBz) + 0.000 1.0306 0, 023C47 0.913032 Beer's Law Curve For Benzoic Acid At 5.023 Microns In Chloroform Concentration - moles per liter Blank Sample Blank 4%T Ave. =e Log ave wae 7 | 06.1 42,3 96.1 44.016 0.3183 96.3 42.2 96.5 43.798 2.2837 6.358643 C6.7 42.1 96.6 43.559 94.5 57.2 C4.2 60.625 0.1912 o5.4 57.3 95.2 60.125 1.6556 ¢.219034 04.8 57.2 94.9 6c.411 62.8 60.6 92.7 65,336 0.1592 93.1 60.5 93.3 64.914 1.5377 0.186766 93.3 €0.5 93.3 64,844 0.1272 03.7 66.0 93.5 70.512 1.4169 0.15308 63.5 66.0 93.4 70,626 92.7 68.5 92.5 73.974 0.1061 92.7 68.5 93.1 73.735 1.3537 0.131524 G2.7 68.5 92.7 73.8S4 92.1 72.9 92.2 79.11C 0.0795 92.2 73.0 92.3 79.132 1.2602 0.10034 91.9 73.1 92.0 79.499 92.0 75.4 92.0 81.556 9.0637 92.2 75.8 92.3 82.168 1.2203 0.08468 $2.3 75.5 92.5 81.709 Log 12 = 1.710(HBz) + 0.020 Beerts Law Curve For Benzoic Acid At 7.570 Microns In Chloroform Concentration moles per liter Blank Sample Blank aay Ave 42 Log ave 4° 66.1 53.5 96.2 55.642 0.3183 65.3 53.6 °5.3 56.243 1.7885 0.25245 O5.4 53.5 95.5 56.050 95.3 60.1 95.2 63.0S7 QO, 25464, O3,4 50,0 93.6 63.101 1.5850 0.20003 93.6 59.1 93.8 63.073 92.8 65.5 93.0 70.505 90,1912 53.0 65.5 03.2 70.354 1.4197 0.152197 02.9 65.3. $2.5 7o0.44e 92.4 68.4 92.0 74.186 0.15615 C1.6 68.1 ©O1.4 74,426 1.3443 6, 128464 O14 68.1 01.3 74.548 A5 78.389 O4,3 78.412 1.2755 0.105680 44.0 78.385 | 0.1273 LOND oan ang UIE OU —~ nod bh LN ELVEN 94.6 81.6 94.8 86.166 0.0795 o4.8 81.7 4.9 86.136 1.1602 6.064534 94.6 81.6 94.6 86.257 05.1 84.2 995.1 88.538 0.0637 o4.5 83.9 $4.5 88.783 1.1278 6.051462 95.6 64.2 94.9 88.678 . Lo . Log “> = 0.7790(HBz) + 0.000 -9O7- Summary Of Data For The Para-Methoxybenzoic-Acetic Anhydride System Principal Wave length A. B. o. p-Methoxybenzoic-acetic mixed anhydride -- 8.711 microns Acetic anhydride ------------------- 8,888 microns p-Methoxybenzoic anhydride --------- 8.1770 microns Beer's Law Curves A. p-Methoxybenzoic-acetic mixed anhydride 1. ie) Cars 3. At mixed anhydride wave length (8.711 microns) Log a. = ,220(M-pMeOBz) + 0.015 At acetic anhydride wave length (8.888 microns) Log 2 = 0.9541 (M-pMeOBz) + 0.0042 At p-methoxybenzoic anhydride wave lengt (8.1770 microns) Log 19 = 0,.1732(M-p-MeOBz) + 0.00225 p-Methoxybenzoic anhydride 1. a ae At mixed anhydride wave length (8.711. microns) - Log + = 0.3945 (p-MeOBz) + 0.000 At acetic anhydride wave length (8.888 microns) Log ae = 0.1885 (p-MeOBz) + 0.000 At p-methoxybenzoic anhydride wave length (8.1770 microns ) Log 2 = .,350(p-MeOBz) + 0.0135 C. Acetic anhydride O = “ Log 4> = 0,.857(Ac) + 0,003 I} 2. At mixed anhydride wave length (8.711 microns) 2, At acetic anhydride wave length (8.888 microns) hog a = 3,40i(Ac) + 0.024 3. p-Methoxyvenzoic anhydride wave length (8.1770 microns} Log 42 = 0,5045(Ac) + 0.002 Simultaneous Eauations Derived From Tne Above Data At 8.711 microns 4 220(M-p-MeOBz) + 0.3945(pMe0Bz) + 0.857(Ac) Log 42 - 0,018 At 8.888 microns 0.954: (M-p-MeOBz) + 0.1885(p-MeOBz) + 3.491(Ac) Log 42 - 0.028 T At 8.1770 microns 0.1732 (M-p-MeOBz) + 4.35(o-MeOBz) + 0.5C45(Ac) Log 42 - 0.018 \ HH Beer's Law Curve For p-Methoxybenzoic Anhydride At 8.7110 Microns In Chloroform 08 KH Concentration moles per liter Blank Sample Blank 4T Ave, LO o7.4 88.9 97.7 91.132 0.0941 07.6 88.9 97.8 90.992 1.0987 97.8 89.0 98.0 96.909 07.2. 90.1 97.3 92.647 0.07528 97.3 90.1 97.2 92.647 1.0795 97.4 90.0 97.0 92.592 97.1 91.8 97.1 94.541 0.05646 97.2 91.9 97.4 oO4.450 1.0582 O7.1 91.7 96.9 94.487 98.5 93.9 98.5 95.329 0.04705 98.5 93.9 98.5 95.329 1.0488 98.5 93.9 98.4 95.378 Log 42 = 0.3945(p-MeoBz) + 0.000 Beer's Law Curve For p-Methoxybenzoic Anhydride At 8.963 Microns In Chloroform Concentration Io moles per liter Blank Sample Blank @%T Ave .=- . 97.6 91.8 97.5 94.105 . O.1414 | 07.6 91.8 S7.6 94.057 1.06038 97.5 91.7 97.4 94.099 97.5 93.0 97.3 95.482 0.10605 07.0 92.7 97.2 95.468 1.0474 97.0 92.5 96.8 95.45¢ 07.4 94.5 97.5 96.972 0.0707 97.5 94.6 97.6 96.975 1.0317 07.6 94.5 97.4 96.923 Log £2 = 0.1888(p-MeoBz) + 0.000 Log ave. 42 0.040880 0.03220 eo Lo Log ave, a 0.025638 0, 020014 0.013554 -100- Beer's Law Curve For p-Methoxybenzoic Anhydride At 8.177 Microns In Chlorotcrm Concentration ' Lo moles per liter Blank Sample Slank ot AV aa Log ave. o7.b 46.60 97.2 47.863 a - } a cy hic a My Met? Fa DIAROVIM 0.0707 o6.6 46.2 96.7 47.630 2.0913 0.320210 S6.7 46.2 96.5 47,826 S46 52.5 94.7 55.407 . 0.0565 o4.6 52.5 S4.5 55.408 2.8043 6.25631¢ G47 52.6 95.0 55.455 95.6 60.6 95.7 63.355 emlpes ti oye for am ats + yo al o a eOor , C4pk 05.6 60.5 $5.7 63.355 1.5803 0,1°8768 95.8 60.5 95.9 63.119 5.7 65.0 65.5 67.991 0.03535 c5.4 64.8 $5.5 67.888 1.4704 0.167436 $5.0 64.9 $5.5 68.136 65.5 69.8 05,2 73.203 —— 0.0283 95.0 69.7 95.2 73.201 1.3653 0.135196 65.1 69.5 C4.7 73.234 o4.8 80.1 95.0 84.404 O.,C1l415 ch.3 76.6 GH.3 B4.Al1 21,1044 Oo, 073498 C4h.5 79.9 94.7 BE .4EC 95.7 85.8 95.5 ¢O.7cC4 0.007075 65.4 86.7 95.7 SO.737 1.1016 c.oke141 05.8 87.0 96.0 SO.7LS trl H5 4 .350(p-MecBz) + 0.0135 } li tt 9 58 ~LO1- Beer's Law Curve For p-Methoxybenzoic-Acetic Mixed Anhydride at &.7110 Microns In Chloroform Concentration moles per liter Blank Sample Blank T Ave 22 _ bog ave 72 | O7-7 24.5 97.7 25.076 0.13980 98.0 24.6 97.6 25.153 3.9836 0.600166 97.5 24.5 97.9 25.076 98.3 32.5 98.5 33.028 0.11140 908.5 32.4 98.4 32.910 3.0329 0.481856 08.3 32.3 98.2 32.875 98.0 38.4 98.0 89.183 0.09320 97.9 38.1 97.8 38.937 2.5583 0.407951 97.9 38.3 97.8 39,141 O7.7 53.8 97.7 56.090 | 0.05592 97.8 54.9 97.8 56.134 1.7843 0.251465 98.1 54.9 98.3 55.906 95.7 58.9 95.9 61.482 0. 04464 96.0 59.0 95.8 61.522 1.6255 ~ 0.210985 95.8 58.9 95.6 61.546 OS . 98.4 72.6 98.1 73.893 0.02796 97.6 72.3 98.0 73.926 1.3531 0.131335 98.3 72.6 98.2 73.893 98.0 85.7 98.2 87.461 0.011160 98.1 85.9 97.9 87.653 1.1414 0.057434 07.9 86.0 98.2 87.710 i o 09 . i =° = .,220(M-p-MeoBz) + 0.015 ~102- Beer's Law Curve For p-Methoxybenzoic-Acetic Mixed Anhydride At 5.536 Microns In Chloroform Concentration To moles per liter Blank Sample Blank ST Ave. 2 Log ave Fr 97.9 73.8 97.6 75.498 0.12140 07.5 73.6 97.6 75.448 1.3265 0.122705 97.6 73.7 97.6 75.512 98.1 77.9 97.8 79.530 0.09712 97.5 77.7 97.7 79.610 1.2600 0.10037 07.7 77.6 S7.4 79.548 97.9 82.6 97.7 84.458 ; 0.07284 97.8 82.6 97.9 83.414 1.1861 0.074117 07.7 82.7 97.7 84.646 98.1 87.0 98.1 88.685 0.04856 98.1 87.1 98.2 88.741 1.1275 0.052115 98.0 87.0 98.3 88.639 98.3 89.7 98.2 91.297 0.03642 98.0 89.4 97.9 91.271 1.0957 0.039697 98.0 89.4 98.0 91,224 Log #2 = 0.9541(M-p-MeoBz) + 0.0042 Beer's Law Curve For p-Methoxybenzoic-Acetic Mixed Anhydride At 8.177 Microns In Chloroform Concentration to moles per liter Blank Sample Blank eT Ave Fe Log aVe > 98.0 87.1 97.9 88.922 0.01368 98.1 87.2 98.1 88.888 1.1245 0.050960 97.8 87.1 98.0 88.968 97.8 88.9 97.6 90.992 . 0.01118 97.5 88.7 97.5 90.974 1.0992 0.041078 67.4 88.5 97.2 90.955 97.0 90.1 96.8 92,982 0.00839 97.5 90.5 97.2 $2.963 1.0764 0.031974 97.0 90.1 97.3 92.748 96.5 90.9 96.5 94.196 0.00699 96.5 90.9 96.5 94.196 1.0606 0.025556 96.4 91.0 96.3 S4.447 ie) 0.1732(M-p-MeoBz) + 0.00225 i 03 H{5' | -103- Beer's Law Curve For Acetic Anhydride At 8.7110 Microns In Chloroform Concentration I To moles per liter Blank Sample Blank %T Ave =2 Log ave. 07.3 40.3 97.4 41.294 oO. 4242 o7.4 Ho. 97.4 42.170 2.4224 0.38422 DS 97.2 40.2 C7.1 41.379 07.4 46.7 97.3 47.971 . 0.3610 07.3 46.8 07.2 48.123 2.0838 0.318858 67.3 46.6 97.0 47.967 97.4 55.5 97.3 57.010 C.2766 O7.3 55.3 97.2 56.834 1.7572 0, 24h820 67.3 55.4 97.5 56.878 96.8 63.3 $6.8 65.392 0,2122 06.6 63.1 96.5 65.354 1.5306 0.184864 96.5 63.0 96.3 65.352 06.7 71.5 96.5 74.016 0.1488 96.5 71.5 96.5 74.093 1.3495 0.130138 s6.4 71.4 96.1 74.181 06.4 77.6 96.4 80.597 0.10561 06.32 77.5 96.4 80.435 1.2418 0.094050 96.2 77.4 96.0 80.541 96.5 84.5 96.6 87.519 0.06375 06.6 84.6 96.6 87.577 1.1424 0.057822 96.6 84.6 $6.8 87.487 96.4 88.0 96.3 $1.333 9.04244 06.4 88.0 96.3 &1.333 1.0060 0.03981 96.5 87.9 96.5 $1,040 Io Ui 0.8570(Ac) + 0.003 -104- Beer's Law Curve For Acetic Anhydride At G.A77 Microns Ta Chioroforn Concentration moles per liter Blank Sample Blank BT Ave Lo Log ave 22 ohne £ 7 OS EVe = S8.4 59.3 98.4 60.264 0, 4248 $8.3 59.2 98.4 60.192 1.6571 0.219346 08.5 56.3 98.4 60.233 08.3 72.1 98.2 73.384 0.2548 98.2 72.1 98.2 73.421 1.3617 0.133764 98.0 72.0 97.9 73.506 98.0 75.8 98.0 77.346 . 0.2122 98.0 75.8 97.6 77.505 1.2603 0.1106¢2 S7.6 75.7 97.4 77.641 98.4 82.0 98.4 83.333 0.1488 98.4 82.0 98.4 83.333 1.1997 0.079072 98.5 82.1 98.4 83,3¢2 O7.7 85.8 S7.4 87.954 0.1061 08.3 86.2 98.2 87.735 1.1396 9.056748 98.3 86.1 98.4 87.545 08.4 °c0.8 98.4 92.276 Q.06375 98.5 91.0 98.5 92.385 1.0771 0.063226 $8.5 90.9 98.7 92.190 Log >> = O.5C45(Ac) + 0.002 --105- Summary Of Data For The Para-Nitrobenzoic-acetic Anhydride System Principal Wave Length A, p-Nitrobenzoic-acetic mixed anhydride --- 7.897 microns B. Acetic anhydride ------------------------ 8.888 microns C. p-Nitrobenzoic anhydride ---------------- 6.486 microns Beer's Law Curves A. p-Nitrobenzoic-acetic mixed anhydride 1. At mixed anhydride wave length (7.897 microns) Log 2 = 3.341 (M-p-Nitro Bz) + 0.000 2, At acetic anhydride wave length (8.888 microns) Log ae = 1.601(M-p-Nitro Bz) + 0.0036 3, At p-Nitrobenzoic anhydride wave length (6.486 microns) Log 32 = 5.30(M-p-Nitro Bz) + 0.004 | B. p-Nitrobenzoic anhydride 1. At mixed anhydride wave length (7.897 microns ) Lo Log = = 0.559 (p-Nitro Bz) + 0.0025 2. At acetic anhydride wave length (8.888 microns) Log 42 = 0,181 (e-Nitro Bz) + 0.000 3. At p-nitrobenzoic anhydride wave length (6.486 microns) Log #2 = 11.09(p-Nitro Bz) + 0.000 -106- C. Acetic Anhydride 1. At mixed anhydride wave length (7.897 microns) Log tO = 0.2535(Ac) + 0.000 2, At acetic anhydride wave length (8.888 microns) Log 42 = 3.491(Ac) + 0.Cc24 3. At p-nitrobenzoic anhydride wave length (6.486 microns) Log a = 0.587(Ac) + 0.002 Simultaneous Eguations Derived From The Above Rata At 7.897 microns 3.341(M-p-Nitro Bz) + 0.559(p-Nitro Bz) + 0.2535(Ac) = Log ae -~ 0.0025 At 8.888 microns 1.601(M-p-Nitro Bz) + 0.181(p-Nitro Bz) + 3.491(Ac) = Log #2 - 0.028 At 6.486 microns 5.30(M-p-Nitro Bz) + 11.09(p-Nitro Bz) + 0,.587(Ac) = Log ze ~ 0.006 -107~ Beer's Law Curve For p-Nitrobenzoic Anhydride At 7.9697 Microns In Chioroform Concentration moles per liter Blank Sample Blank 4T Ave #0 Log ave.50 98.9 93.7 98.5 S4.934 0.0381 08.7 93.6 98.6 94.880 1.0535 0.022635 98.6 953.7 98.8 94.934 98.1 93.4 97.8 95.354 0.03422 98.4 93.5 98.3 95.068 1.04902 0.020846 97.8 93.3 97.6 95.496 98.9 95.6 98.9 96.663 0.02285 98.7 95.5 98.5 96.855 1.0336 0.014352 98.8 95.6 98.9 96.713 98.9 97.0 98.9 98.078 0.01123 98.8 97.0 98.9 98.128 1.0194 0,008346 98.9 97.0 98.9 98.078 Log 42 = 0,5590(p-Nitro Bz) + 0.0025 Beer's Law Curve For p-Nitrobenzoic Anhydride At 3,605 Microns In Chloroform Concentration r . To moles per liter Blank Sample Blank eT Ave .=2 Log ave. 98.6 94.3 98:8 95.542 | 0.0960 98.9 94.4 98.7 S5.546 1.0468 0.019866 98.9 S4.3 98.8 95.493 98.6 96.5 98.7 97.820 0.0480 98.5 96.4 98.6 97.818 1.0221 0.009493 98.4 96.3 98.4 97.865 98.4 96.7 98.6 98,172 0.0384 98.6 96.7 98.32 98.222 1.0168 0.007234 98.4 96.7 98.3 98,322 Log 42 = 0,1810(p-Nitro Bz) + 0.000 -108- Beerts Law Curve For p-Nitrobenzoic Anhydride At 6.486 Microns In Chioroform Concentration moles per liter Blank Sample Blank 4T Ave . 40 Log ave. 98.2 45.1 98.1 45.950 0.03060 98.5 45.2 98:4 45.911 2.1915 0.340740 98.1 45.1 98.3 45.926 68.5 54.9 98.7 55.679 0.02295 S8.9 55.0 S8.6 55.724 1.7952 0.254110 98.3 54.7 98.1 55.702 97.7 66.2 97.6 67.793 6.01530 97.7 66.2 97.5 67.827 1.4755 0.168S40 97.5 66.1 97.8 67.690 97.5 77.0 97.3 79.055 0.00922 97.4 77.0 97.3 79.096 1.2641 0.101784 97.3 76.9 97.0 79.155 97.5 80.4 97.5 82,461 0.00765 07.7 80.4 97.6 82.334 1.2142 c0.084288 97.8 80.5 97.9 82.268 07.2 83.3 97.1 85.743 0.00614 97.1 83.3 97.2 85.743 1.1668 0.066996 97.3 83.4 97.5 85.626 97.1 89.9 97.1 92.584 0.00307 97.2 89.9 97.1 92.537 1.0808 0.033748 97.3 90.0 97.4 92,449 Log 22 = 11.09(p-Nitro Bz) + 0.0000 -109- Beer's Law Curve For p-Nitrobenzoic-acetic Mixed Anhydride At 7.397 Microns In Chloroform Concentration moles per liter Blank Sample Blank @T Ave 19 Log ave.72 -90.8 49.8 91.1 54.755 0.0783 91.0 49.8 91.2 54,665 1.8309 0.262466 91.2 49.8 90.9 54.426 91.2 56.3 91.4 61.664 0.06264 91.3 56.2 92.0 61.656 1.6223 0.212531 91.3 56.3 91.5 61.597 89.9 65.3 90.0 72.595 0.0426 90.1 65.2 89.9 72.444 1.3819 0.140168 90.1 65.2 90.0 72.044 92.3 68.7 92.1 74,511 0.3915 92.1 68.7 92.5 74.431 1.3429 ©.128047 92.5 68.9 92.6 74.446 90.4 74.1 90.2 82.059 0.02555 90.2 74.4 90.4 82,392 1.2172 0.085362 90.4 74.1 90.3 82.014 90.4 78.8 90.3 87.216 0.01704 90.3 78.8 90.1 87.361 1.1445 6,058763 00.1 78.7 89.9 87.444 98.0 91.5 97.9 93.415 0.00852 98.1 91.5 97.9 93.319 1.0710 0.02979 97.9 91.6 98.3 93.374 Lo = 3,341(M-p-Nitro Bz) + 0,000 ~1i0- Beer's Law Curve For p-Nitrobenzoic-acetic Mixed Anhydride At 8.888 Microns In Chloroform Concentration moles per liter Blank Sample Blank 4%T Ave 40 97.8 81.2 97.7 83.069 0.0475 97.6 81.1 97.5 83.136 1.2027 97.6 81.1 97.3 83.222 97.4 83.8 97.0 86.213 0.0380 . 97.0 83.8 97.0 86.391 1.1585 97.1 83.8 97.0 86.347 O7.1 . 86.8 97.1 89.392 0.0285 97.2 86.5 97.0 89.083 1.1206 97.1 86.6 97.0 89,232 97.1 89.8 97.1 92.481 0,0190 97.1 89.7 97.0 92.426 1.0815 97.0 89.8 97.2 92.481 97.1 92.9 97.1 95.674 0.0095 97.1 93.0 97.0 95.826 1.0442 97.0 93.0 97.2 95.777 Log 42 = 1.601(M-p-Nitro Bz) + 0.0036 Beer's Law Curve For p-Nitrobenzoic-acetic Anhydride At 6.400 Microns In Chloroform Concentration moles per liter Blank Sample Blank @T Ave. 40 | 97.1 52.6 97.1 54.170 : 0.0480 97.3 52.7 97.3 54.162 1.8475 97.5 52.8 97.5 54.153 96.5 58.6 96.6 60.693 0.0384 96.4 58.6 96.8 60.662 1,6481 96.7 58.7 96.8 60.671 98.2 67.4 98.2 68.635 0.0288 98.2 67.4 98.1 68.670 1.4562 98.0 67.3 97.9 68.708 98.2 75.9 98.1 77.330 0.0192 98.1 75.9 98.1 77.370 1.2932 98.0 75.8 98.2 77.268 908.1 85.9 98.0 87.608 0.0096 98.0 85.9 98.0 87.653 1.1416 98.2 85.9 98.1 87.519 Log LO = 5.300(M-p-Nitro Bz) + 0.004 Log ave.== Log ave 42 9.080159 0.063895 0.049454 0.034030 0.018784 To C. 266585 0.216986 0.16220 0.111666 0.057134 -lli- Beer's Law Curve For Acetic Anhydride At 7.897 Microns In Chloroform Concentration moles per liter Blank Sample Blank 98. 0.5244 98. 98. 38. 0.3486 98. 98. 98. 0.2622 98. 98. 98. 0.1748 98. 98. 98. 0.1049 9 98. NNO NFU OVCOCO FOM NOVO 73. C 72.9 72. 80. 80. 80. 84, 84, 84, 88. 88. 88, 92, 92. 92. NO} Hoe CON CO NON OWO 6T 98.5 73.924 98.7 73.897 98.8 73.822 98.5 81.443 98.8 81.477 98.2 81.609 98.9 85.887 98.5 86.061 98.7 85.960 98.5 90.050 98.7 89.903 98.6 89.913 98.8 93.927 98.7 93.920 98.8 93.974 0.2535(Ac) + 0.0000 Ave. Lo _—_— wi I 1.3535 1.2268 1.1632 1.1116 1.0645 Beer's Law Curve For Acetic Anhydride At 6.486 Microns In Chloroform Concentration moles per liter Blank Sample Blank 4%T 98.1 91.0 98.1 92.762 0.5244 98.1 91.1 98.2 92.817 98.2 91.0 98.0 92.762 98.1 G2.0 98.1 93.781 0.4370 98.1 92.0 98.0 93,829 98.1 92.1 98.3 93.788 97.9 93.0 97.9 94.994 0.3486 97.9 93.0 97.9 94,994 97.9 93.0 $7.9 94.994 97.8 93.4 97.7 95.549 0.31464 97.8 93.3 97.6 95.496 97.6 93.3 97.7 95.545 Lo Log T 0.5870(Ac) + 0.002 Lo 1.0778 1.0661 1.0526 1.0467 Log ave.42 0.13146 0.088500 C.065654 0.045956 0.027145 Log ave 42 0.032540 0.027800 0.022225 9,019824 -lle- Equilibrium Mixture Derived From Acetic Anhydride And Benzoic Acid In Chloroform A€ 150°C Wave length Equilibriun Constant (acid-II) ---- 6. (microns) Blank Sample Blank #T Average #2 Log ave 20 | 96.8 45.4 66.9 46.876 8.690 97.0 45.5 ¢67.2 46.858 2.1333 0.32905 97.3. 45.6 97.2 46.889 oo 96.0 44.8 S6.1 46.642 8,888 96.2 Hb.g 96.2 46.673 2.1433 0.33108 96.5 45.0 96.4 46.656 96.5 58.5 96.7 60.559 8.224 06.7 58.6 96.9 60.537 1.6518 0.217958 96.9 58.7 97.1 60.515 96.0 63.0 96.2 65.556 5.823 96.3 63.1 96.2 65.558 1.5256 0.183438 96.2 63.1 96.4 65.524 95.9 84.4 96.2 87.870 7.570 96.0 84.3 95.9 87.858 1.1386 0.056368 95.9 84.2 95.8 87.845 Concentration benzoic-acetic mixed anhydride -- 0.1213350 Concentration bénzoic anhydride ----~---------- O.0433680 Concentration acetic anhydride ----~-~---------- 0. 0686636 Concentration benzoic acid ---------~-~-----~--- O,0121030 Concentration acetic acid --~-------~-------~--- 0.0437800 Equilibrium Constant (anhydride)--- 4.94 Equilibrium Constant (acid-I) ----- 1.29 -113 Equilibrium Mixture Derived From Acetic Anhydride, 6 Benzoic Anhydride And Acetic Acid In Chloroform At 75°C Wave length Lo (microns) Blank Sample Blank @T Average ie Log ave .=> SG7.6 67.0 97.8 68.5772 8.690 97.5 66.9 97.6 68.5802 1.45843 0.163881 97.8 67.0 97.7 68.5421 : 97.8 63.8 97.9 65.2018 8.888 07.8 63.7 97.6 65.1995 1.53375 © 0.185755 97.6 63.6 97.5 05.14/53 y7 4 HO.6 97.3 50.9501 8.224 97.5 49.7 97.6 50.9482 1.96277 0.292874 97.8 49.8 97.7 50.9462 $7.8 57.1 98.0 58.3248 5.823 98.1 57.2 98.2 58.2781 1.71528 0.234338 98.0 57.1 97.9 58.2650 97.9 84.7 97.9 86.5168 7.570 97.8 84.6 97.7 86.5473 1.15557 0.062601 97.7 84.6 97.8 86.5473 Concentration benzoic-acetic mixed anhydride -- 0.056873 Concentration benzoic anhydride ----------~----- 0.0721210 Concentration acetic anhydride ---------------- 0.038472 Concentration benzoic acid -------------------- 0,0418791 Concentration acetic acid -----~---------------- 0.0520808 Equilibrium Constant (anhydride) -- 1.25 Equilibrium Constant (acid-I) ----- 1.58 Equilibrium Constant (acid-II) ---- 1.97 -114- Bgquilibrium Mixture Derived From Benzoic-acetic Mixed Anhydride And Benzoic Acid In Chloroform At 75°C Wave length (microns) Blank Sample Blank %T Average 4° Log ave 72 95.7 56.5 95.8 59.0078 8.6909 95.8 56.6 95.9 59.0505 1.66436 0.229013 95.9 56.6 96.0 58.9890 96.1 46.4 96.3 48.2077 8.888 96.5 46.5 96.5 48.1865 2.07711 0.317462 96.4 46.5 96.4 48.2365 S6.7 50.1 96.5 51.8633 8,224 96.5 50.1 96.6 51.8902 1.92662 0.285470 96.6 50.1 96.4 51.9170 96.3 48.7 96.2 50.5474 5,823 96.1 48.6 96.0 50.5986 1.97671 0.295834 06.2 48.7 96.4 50.5711 96.4 83.1 96.5 86.1586 7.570 96.5 83.2 96.6 86.1729 1.16092 C.064-704 96.6 83.2 96.7 86.0838 Concentration benzoic-acetic mixed anhydride -- 0,0752055 Concentration benzoic anhydride --------------- -0.0738982 Concentration acetic anhydride ---------------- C.0708808 Concentration benzoic acid -------------------- O.0250481 Concentration acetic acid --------------------- ‘0,0857286 Equilibrium Constant (anhydride) - 1.08 Equilibrium Constant (acid-I) ---- 3.36 Equilibrium Constant (acid-II) --- 3.61 -115- Equilibrium Mixture Derived From Benzoic-acetic Mixed Anhydride And Benzoie Acid At 100°0C In Ghlioroform Wave Zength (microns) Blank Sample Blank 4%T Average 49 Log ave .40 ; O7.3 56.7 97.2 58.303 8.690 97.5 56.8 97.3 58.316 1.7153 0.234338 97.2 56.7 97.4 58.273 96.8 53.4 96.9 55.136 8.888 96.7 53.2 96.5 55.072 1.8149 0.258859 96.4 53.1 96.4 55.082 96.3 51.4 96.2 53.402 8, 22k 96.1 51.4 96.1 53.485 1.8709 0.272047 96.1 51.4 96.2 53.458 96.5 AM.7 96.4 46.345 oo 5.823 96.4 Ah.7 96.4 46.369 2.1561 0.33367C 96.5 44.8 96.5 46,424 95.8 80.4 96.0 83,837 ; 7.570 96.0 80.5 95.9 83.897 1.1922 0.076352 95.9 80.4 95.8 83.881 Concentration benzoic-acetic mixed anhydride -- 0.0829080 Concentration benzoic anhydride --~------------- 0.0663150 Concentration acetic anhydride ---------------- 0.0517563 Concentration benzoic acid ~------------------- 0.0408189 Concentration acetic acid ---------------~------ 0.0946918 Equilibrium Constant (anhydride) -- 2.00 Equilibrium Constant (acid-I) ----- 1.86 Equilibrium Constant (acid-II) ---- 2,04 -116-~- Equilibrium Mixture Derived From Benzoic Anhydride And Acetic Acid At 100°C In Chloroform Wave length (microns) Blank Sample Blank 47 Average 10 Log ave 4° 96.7 45.2 96.8 46.718 8.690 96.8 45.2 96.9 46.670 2.1409 0.330590 96.9 45,2 96.7 46.743 95.9 47.2 96.0 49.192 8,888 95.9 47.1 95.8 49.139 2.0339 0.308329 95.8 47.1 95.8 49.164 95.4 4e.,e 95.6 44,188 8. 224 95.6 42.2 95.5 44.165 2.2651 0.355079 95.6 42.2 95.7 44.119 95.7 43.5 95.7 45.454 Oo 5.823 95.8 43.6 96.1 45.393 2.2001 0.342439 96.0 43.7 96.0 45.520 96.1 79.3 96.2 82.475 7.570 96.3 79.6 96.5 82.572 1.2119 0.083464 96.5 79.6 96.5 82.487 Concentration benzoic-acetic mixed anhydride -- 0.122560 Concentration benzoic anhydride -------------+- 0.082450 Concentration acetic anhydride ---------------- 0.058464 Concentration benzoic acid -------~--~---~------- 0,029669 Concentration acetic acid ----------~----~------ 0.098309 Equilibrium Constant (anhydride) -- 3.12 Equilibrium Constant (acid-I) ----- 2.23 Equilibrium Constant (acid-II) --~-- 6.95 -117- Equilibrium Mixture Derived From Benzoic-acetic Mixed Anhydride And Acetic Acid At 15000 Im Chioroform Wave length (microns) Blank Sample Blank %2 Average 20° Log ave 20 . 97.8 65.4 97.8 66.871 8.690 97.7 65.2 97.4 66.837 1.4956 0.174814 97.4 65.1 97.3 66.872 97.5 63.3 97.4 64.959 8,888 97.3 63.2 97.5 64.887 1.5403 0.287604 97.5 63.3 S7.5 64.923 97.4 1.2 67.3 73.138 8,224 97.1 1.1 97.3 73.148 1.3675 0.13583 97.3 7i.1l 97.0 73.185 97.0 56.3 96.9 58.071 5.823 96.9 56.2 96.7 58.057 1.7230 0.23629 96.7 56.1 96.8 57.984 97.3 86.9 97.2 89.357 7.570 97.2 86.9 97.2 89.403 1.1191 0.048860 97.3 86.9 97.3 89.311 Concentration benzoic-acetic mixed anhydride ~-- 0.06296500 Concentration benzoic anhydride --------------- 0.02781174 Concentration acetic anhydride ---------------- 0.03813330 Concentration benzoic acid -------------- ae 0,02406000 Concentration acetic acid ---~-~----~------~----~- 0.07184750 Equilibrium Constant (anhydride) -- 3.74 Equilibrium Constant (acid I) ----- 1.27 Equilibrium Constant (acid II) ---- 4.75 -118- Equilibrium Mixture Derived From Benzoic And Acetic Anhydrides At 70° In Chloroform Wave Length Blank Sample Blank eT Average $T Log ave .42 96.8 73.9 26-7 76.382 8.224 96.7 73.9 96.8 76.382 76.395 0.116907 96.7 73.9 96.7 76.421 97.7 59.7 97.8 61.074 8.888 97.5 59.6 $7.7 61.056 61.078 0.214102 97.6 59.6 97.5 61.096 96.5 57.5 96.4 59.512 8.690 96.4 57.4 96.3 59.574 59.564 0.224998 96.3 57.4 96.3 59.605 Concentration of benzoic-acetic mixed anhydride - 0.0818490 Concentration of benzoic anhydride -------------- 0.0018287 Concentration of acetic anhydride -~------------- 0.0352680 Equilibrium Constant = 1.04 Equilibrium Mixture Derived From Benzoic-Acevic Mixed Anhydride At 70° C In Chloroform Wave Length Blank Sample Blank oT Average %T Log ave 4? 95.4 66.6 95.5 69.774 8,224 95.6 66.8 95.7 69.837 69.772 0.156340 95.4 66.5 95.4 69.706 94.8 58.2 95.0 61.327 8.888 94.7 58.1 94.6 61.384 61.362 0.212182 94.5 58.0 94.5 61.375 . 95.8 52.7 96.0 54.953 8.690 96.1 52.8 96.1 54.942 54.955 0.259974 96.0 52.8 96.1 54.971 Concentration of benzoic-acetic mixed anhydride -- 0.0972660 Concentration of benzoic anhydride --------------- 60,0281 644 Concentration of acetic anhydride --~-------------- 0.031366C Equilibrium Constant = 1.07 -119- Equilibrium Mixture Derived From Benzoic And Acetic Anhydrides At 100°C In Chloroform Wave Length Blank Sample Blank %T Average ZT Log ave Lo I 94.8 68.1 94.9 71.797 8,228 95.0 68.3 95.2 71.819 71.799 0.143733 95.2 68.3 95.1 71.781 05.4 67.9 95.5 70.193 8,888 95.5 67.0 95.5 70.157 70.192 9.153660 95.6 67.1 95.5 70.225 c6.9 55.9 S6.1 58.198 8.690 C6.0 55.9 $5.9 58.259 58,220 0.234845 S6.0 55.9 96.0 58.225 Concentration of benzoic-acetic mixed anhydride - 0.0911480 Concentration of benzoic anhydride -~------------- 0.0265109 Concentration of acetic anhydride --~---~---~------ 0.0157940 Equilibrium Constant = 1.98 Equilibrium Mixture Derived From Benzoic-Acetic Mixed Anhydride At 100°¢C In Chloroform Wave Length Blank Sample Blank %T Average @T Log ave ae 96.0 38.3 96.2 39.854 © 8,224, 96.3 38.4 96.4 39.853 39.826 0.399836 96.3 38.3 96.3 39.771 97.8 35.8 °7.8 36.605 | 8.888 97.6 35.8 97.7 36.661 36.630 0.4361600 97.8 35.8 97.7 36.624 96.5 43.0 96.4 44,5802 8.690 96.5 43.1 96.6 44.640 44.605 0.350620 06.6 43.2 96.7 44.593 Concentration of benzoic-acetic mixed anhydride - 0.120114 Concentration of benzoic anhydride -------------- 0.088116 Concentration of acetic anhydride ---------~--~--- 0.088350 Equilibrium Constant = 1.85 -120- Equilibrium Mixture Derived From Benzoic And Acetic Anhydrides At 150°C In Chloroform Wave Length Blank Sample Blank @%T Average @T Log ave 22 L . 95.6 63.4 95.7 66.283 822k 95.4 63.3 95.6 66.282 66.294 0.178516 95.5. 63.3 95.4 66.317 ; . | 94.8 52.5 94.7 55.408 8.888 O4.7 52.5 94.9 55.379 55.391 0.256552 95.0 52.6 94.9 55.397 DO 96.7 56.3 96.8 58.191 8.690 96.9 56.4 96.9 58.204 58.210 0.234995 96.8 56.4 96.9 58.234 Concentration of benzoic-acetic mixed anhydride - 0.0826050 Concentration of benzoic anhydride -------------- 0.0339594 Concentration of acetic anhydride --------------- 0. 0466070 Equilibrium Constant = 4.31 Equilibrium Mixture Derived From Benzoic-Acetic Mixed Anhydride At 150°C In Chloroform Wave Length Blank Sample Blank GL Average %T Log ave AP . 96.4 57.5 96.3 59.678 8,224 | 96.3 57.5 96.3 59.709 59.709 0.223935 96.3 57.5 96.2 59.740 95.7 51.6 95.8 53.890 8.888 © 96.1 51.9 96.2 53.978 53.946 0.268041 96.3 52.0 96.4 53.969 95.4 52.0 95.4 54.507 8.690 95.5 52.1 95.5 54.554 54.529 0.263352 95.6 52.1 95.5 54.526 | Concentration of benzoic-acetic mixed anhydride - 0.094565 Concentration of benzoic anhydride -------------- 0.045713 Concentration of acetic anhydride --~------------ 0.046949 Equilibrium Constant = 4.17 ~l2i- Equilibrium Mixture Derived From p-Methoxybenzoic And Acetic Anhydrides At 75°C In Chloroform Wave Length Blank Sample Blank er Average @T Log ave 42 . 97.0 62.6 97.1 64.502 8.711 97.2 62.7 97.1 64.539 64.537 0.190162 97.1 62.7 97.1 64.572 oe 98.1 70.8 98.2 72.134 8,888 98.0 70.7 97.9 72.179 72.155 0.141729 o7 .9 70.6 97.8 72.151 97.5 72.0 97.6 73.808 8.177 97.7 72.1 97.8 73.759 73.720 | 0.132068 97.7 72.0 S7.7 73.694 Concentration of p-methoxybenzoic-acetic mixed anhyd - 0.0342403 Concentration of p-methoxybenzoic anhydride -------~--- 0.0223059 Concentration of acetic anhydride ----------------~--.- 0, 0220160 Equilibrium Constant = 2.26 Equilibrium Mixture Derived From p-Methoxybenzoic Acetic Mixed Anhydride At 75°C In Chloroform Wave Length Blank Sample Blank oT Average @T Log ave 42 95.8 46.8 95.9 48.826 8.711 95.9 46.7 95.7 48.747 48.790 0.311666 95.7 46.7 95.7 48.798 94.9 57.0 95.0 60.031 . 8.888 95.2 57.1 95.1 60.010 60.003 0.221805 95.0 57.0 95.1 59.968 96.0 58.2 96.2 60.561 8.177 96.2 58.3 96.3 60.571 60.554 0.217854 96.2 58.3 96.4 60.539 Concentration of p-methoxybenzoic-acetic mixed anhyd - 0.058300 Concentration of p-methoxybenzoic anhydride ---------- 0.039276 Concentration of acetic anhydride -~-------~-------~---- 0.037472 Equilibrium Constant = 2.31 -~12e- Equilibrium Mixture Derived From p-Methoxybenzoic And Acétic Anhydrides At. 75°c In Chlorororm Wave Length Blank Sample Blank oT Average @T Log ave 42 97.2 33.5 97.3 34.465 8.711 97.5 33.6 97.5 34.461 34.456 0.462730 S7.6 33.6 97.5 34.443 S7.9 35.0 97.8 35.769 8,888 97.8 35.0 97.8 35.787 35.787 0.446275 97.7 35.0 97.8 35.805 96.0 57.8 96.1 60.176 8.177 96.1 57.8 96.1 60.145 60.179 0.220452 96.2 57.9 96.1 60.218 Concentration of p-methoxybenzoic-acetic mixed anhyd - 0. 083015 Concentration of p-methoxybenzoic anhydride ---------- 0.032177 Concentration of acetic anhydride -------------------- 0.095341 Equilibrium Constant = 2.25 Equilibrium Mixture Derived From p-Methoxybenzoic And Acetic Anhydrides At 100°C In Chloroform Wave Length Blank Sample Blank @T_ Average @T Log ave 42 98.0 57.3 98.1 58.439 8.711 98.2 57.4 98.1 58.481 58.477 0.233000 98.1 57.4 98.1 58.511 97.5 66.8 97.6 68.477 8.888 97.4 66.7 97.5 68.445 68.465 0.164530 97.9 67.0 97.8 68.472 96.0 73.7 96.2 76.69C 8.177 96.3 73.9 96.4 76.699 76.683 0.115280 96.4 73.9 96.4 76.659 Concentration of p-methoxyoenzoic-acetic mixed anhyd - 0, 0440581 Concentration of p-methoxybenzoic anhydride ---------- 0.0O175770 Concentration of acetic anhydride -------------------- 0.0261400 Equilibrium Constant = 4,22 -123- Equilibrium Mixture Derived From p-Methoxybenzoic And Acetic Anhydrides At 100°C In Chloroform Wave Length Blank Sample Blank @T Average @T Log. ave A 94.8 60.6 94.9 63.890 8.711 95.0 60.7 95.1 63.861 63.872 0.194678 95.2 60.8 S5.2 63.865 ; 95.8 65.1 95.9 67.918 8.888 96.0 65.2 96.0 67.916 67.940 | 0.167850 96.1 65.3 96.0 67.985 97.0 81.2 $7.0 83.711 8.177 07.9 81.3 97.1 83.771 83.751 - 0.076892 97.1 81.3 S7.0 83.771 Concentration of p-methoxybenzoic-acetic mixed anhyd - 0.0349620 Concentration of p-methoxybenzoic anhydride ----~--~--- 0.0086781 Concentration of acetic anhydride -------------------- 0. 0300000 Equilibrium Constant ~ 4.6¢ Equilibrium Mixture Derived From p-Methoxybenzoic- Acetic Mixed Anhydride At LOOYC In Chloroform Wave Length Blank Sample Blank %T Average T Log ave.io T 96.4 56.3 96.3 58.432 8.711 95.8 56.0 95.8 58.455 58.451 0.233200 95.9 56.1 96.0 58.467 O4.8 66.9 94.8 70.569 8,888 94.9 67.0 94.9 70.600 70.590 0.151246 94.9 = =67.0 94.9 70.600 95.3 71.0 95.2 74,540 , 8.177 94.9 70.8 95.0 74.565 74.566 0.127430 95.0 70.9 95.1 74.592 Concentration of p-methoxybenzoic-acetic mixed anhyd - 0.044280 Concentration of p-methoxybenzoic anhydride ---------- 0.020830 Concentration of acetic anhydride --------------=----- 0.022085 Equilibrium Constant = 4.26 ~124- Equilibrium Mixture Derived From p-Methoxybenzoic And Acetic Anhydrides At 150°C In Chloroform Wave Length Blank Sample Blank $T Average T Log ave 50 98.0 72.7 98.2 74.108 8.711 98.4 72.9 98.3 74.123 74,329 0.128819 97.9 72.6 97.9 74.157 $6.3 74.4 96.2 77.298 8.888 96.4 74,6 96.5 77.345 77.319 0.111699 96.5 74.6 96.5 77.305 95.8 69.1 95.6 72.280 8.177 95.9 69.2 95.9 72.158 72.205 . 0.141574 96.1 69,4 96.2 72.178 Concentration of p-methoxybenzoic-acetic mixed anhyd - 0.022783 Concentration of p-methoxybenzoic anhydride ---------- 0, 025524 Concentration of acetic anhydride -~--~-~------------- 0.017042 Equilibrium Constant = 11.93 Equilibrium Mixture Derived From p-Methoxybenzoic- Acetic Mixed Anhydride At 150°C In Chloroform Wave Length Blank Sample Blank 4T Average 4? Log ave 52 97.5 61.5 97.4 63.044 8.711 97.3 61.3 97.2 63.033 63.043 0.200356 97.4 61.4 97.3 63.071 98.0 75.5 S7.9 77.080 8,888 97.5 75.1 97.5 77.025 77.072 0.113032 97.7 75.3 97.6 77.112 96.4 78.6 96.4 81.535 8.177 96.7 78.8 96.6 81.531 81.548 0.088560 96.4 78.6 96.3 81.577 Concentration of p-methoxybenzoic-acetic mixed anhyd - 0.039340 Concentration of p-methoxybenzoic anhydride ---------- 0,023158 Concentration of acetic anhydride -------------------- 0,012894 Equilibrium Constant = 9.13 -~125- Equilibrium Mixture Derived From p-Nitrobenzoic And Acetic Anhydrides At 75°C In Chloroform Wave Length Blank Sample Blank %T Average @T Log ave .F0 96.8 65.5 906.9 67.630 7.897 06.7 65.4 96.7 67.422 67.549 0.170380 96.7 65.4 96.8 67.596 98.4 18.8 98.4 19.105 8,888 98.5 18.8 98.6 19.076 19.076 0.720168 98.7 18.8 98.7 19.047 97.8 42,2 97.9 43.127 6.486 66.9 41.8 96.8 43.159 43.156 0.364048 96.8 41.8 96.8 43.181 Concentration of p-nitrobenzoic-acetic mixed anhyd - 0,0354960 Concentration of p-nitrobenzoic anhydride ---------- 0.0057885 Concentration of acetic anhydride ------~----------- 0.1816900 Equilibrium Constant = 1.20 Equilibrium Mixture Derived From p-Nitrobenzoic- Acetic Mixed Anhydride At 75°C In Chloroform Wave Length Blank Sample Blank 47 Average 4 61 Log ave. 42 95.2 76.3 95.3 80,104 7.897 95.1 76.2 95.0 80.168 80.119 0.096244 95.1 76.2 95.2 80.084 97.4. 70.8 97.6 72.615 8.888 G7 .2 70.7 97.3 72.699 72.665 0.138651 97.5 70.9 97.6 72.680 98.1 41.6 97.9 42,448 6.486 97.5 AL.4 97.7 4e.418 42,438 0.372026 98.0 41.6 98.0 42,448 Concentration of p-nitrobenzoic-acetic mixed anhyd - 0.0230540 Concentration of p-nitrobenzoic anhydride --~-------- 0.0209410 Concentration of acetic anhydride --~----~----------- 0.0200378 Equilibrium Constant = 1.27 -126- Equilibrium Mixture Derived From p-Nitrobenzoic And Acetic Anhydrides At 100°C In Chloroform Wave Length Blank Sample Blank %T Average @T Log ave 42 97.1 85.3 97.2 87.802 7.897 96.8 85.0 96.8 87.809 87.815 0.0564060 96.9 85.2 97.1 87.835 an 95.8 78.8 95.9 82.211 8,888 96.0 79.0 96.2 82.206 82.214 —§ 0.0845058 $6.1 79.1 96.3 82,224 97.8 58.0 97.8 59.304 6 486 97.8 58.1 98.0 59.346 59.338 - 0.226652 97.8 58.0 97.6 59.365 Concentration of p-nitrobenzoic-acetic mixed anhyd - 0.0129194 Concentration of p-nitrobenzoic anhydride ---------- 0.0130697 Concentration of acetic anhydride ------------------ 0.00987 00 Equilibrium Constant = 1.29 Equilibrium Mixture Derived From p-Nitrobenzoic- Acetic Mixed Anhydride At 100°C In Chloroform Wave Length Blank Sample Blank %T Average @T Log ave 50 95.6 64.9 95.7 67.851 7.897 095.7 65.0 95.7 67.920 67.898 0.168113 95.5 64.9 95.6 67.922 95.6 47.5 95.6 60.146 8.888 $5.4 57.4 95.6 60.421 60,234 0.220136 95.4 57.4 95.5 60.136 94.8 21.3 94.9 22,456 6.486 95.0 21.3 95.2 22.397 22 417 0.649470 95.1 21.3 95.1 22,397 Concentration of p-nitrobenzoic-acetic mixed anhyd - 0.040816 Concentration of p-nitrobenzoic anhydride ---------- 0.036704 Concentration of acetic anhydride -------~---------- 0.034427 Equiliorium Constant = 1,32 -~127- Equilibrium Mixture Derived From p-Nitrobenzoic And Acetic Anhydrides At 100°C In Chloroform Wave Length Blank Sample Blank %T Average &T Log ave .i2 07.6 76.9 97.5 81.906 7.897 S7.4 79.8 97.4 81.930 81.920 0.086612 97.7 80.0 97.6 81.925 95.6 69.6 95.4 72.879 8.888 95.8 69.8 95.7 72.898 72.869 0.137446 95.5 69.7 95.9 72.831 94.8 47.6 94.7 50.237 6.486 G4.3 47.4 O45) «650.211 50,227 0.299048 96.0 48.2 95.9 50.234 Concentration of p-nitrobenzoic-acetic mixed anhyd - 0.0210147 Concentration of p-nitrobenzoic anhydride ---------- 0.0152745 Concentration of acetic anhydride -------~---------- O,0209212 Equilibrium Constant = 1.38 -128- Equilibrium Mixture Derived From p-Nitrobenzoic And Acetic Anhydrides At 150°C In Chloroform Wave Length Blank Sample Blank ~T Average %T Log ave 52 96.8 71.6 96.8 73.966 | 7.897 96.8 71.6 96.7 74.005 73.979 0.130884 96.8 71.6 96.8 73.966 95.7 61.2 95.5 64.016 8.888 95.6 61.3 95.7 64.087 64.041 © 0.193512 95.7 61.3 95.8 64.026 95.2 36.0 95.3 37.795 6.486 95.3 36.0 95.3 37.775 37.749 0.423080 95.4 35.7 95.5 37.777 Concentration of p-nitrobenzoic-acetic mixed anhyd - 0. 033568 Concentration of v-nitrobenzoic anhydride ---------- 0.020507 Concentration of acetic anhydride ------------------+ 0.030951 Equilibrium Constant = 1.77 Equilibrium Mixture Derived From v-Nitrobenzoic- Acetic Mixed Anhydride At 150°C In Chloroform Wave Length Blank Sample Blank eT Average @T Log ave 22 95.4 79.9 95.3 83.796 7.OS7 95.7 80.3 95.8 83.864 83.841 0.078748 95.8 80.3 95.7 83.864 Ss6.4 74.4 96.4 77.178 8.888 66.5 74.4 96.4 77.138 77.135 0.112742 96.3 74.2 96.2 77.090 $7.3 51.1 97.5 52.464 6.486 CT .7 51.3 97.6 52.534 52 44S C,281076 97.8 51.3 97.7 52.348 Concentration of p-nitrobenzoic-acetic mixed anhyd - 0.019224 Concentration of p-nitrobenzoic anhydride ---------- 0.014840 Concentration of acetic anhydride --------~---------- 0.014690 Equilibrium Constant = 1.70 -12 Q- Rate Of Disproportionation Of p-Methoxybenzoic-acetic Mixed TL (min. )Blank Sample Blank Anhydride At 100°C Reaction Followed By Measurement Of me p-Methoxybenzoic Anhydride 10 15 20 25 30 35 AO Sp | 98. 0 98. o7. 98. 98. 98. 08. 98. 98. 98. 98. O7. o7. OT. 97 . OT. oT. O7. OT. oT. 97. 96. OT. 96. 56. G * 96. ecific 91. Ql. 91. 89. 89. 89. PMO WHF NOW NON RPWOT UOW Oon co an “NIOCMO WOO oO Wn Coun weEwW OND WWW UDF RMPNMO FRW Fru OVON CO 68, 98. og. 98. 98. 98, 98. Rate Constant FOO WOHN WO WFEW WN WOrvouw NOF DOO NFW aa 93 93 93 90. 90. 90. 642 -704 86. 86 86 82 82 82 79 79. 79. 78. 78 78 13 73 73 71. 71. fi. 67. 67. 68. 1. 435 421 421 90S 715 715 704 .961 899 924 425 467 527 376 417 417 .684 .803 . 868 590 509 553 g4e g4e2 151 eg Ave 0° Log ave. Lo 1.0703 1.1015 1.1536 1.2058 1.2582 1.2754 1.3552 1.3976 1.4703 Xx 1073 moles 0.029503 0.041995 0.06206 0.08127 0.09785 0.105646 0.132004 C.145386 — 0.167407 Concentration7+ min. - (liters.mole7+) 8, 8261 9.0334 9.4073 9.7656 16.1210 10.5150 10.928 11.286 11.904 ~t Liters ~130- Rate Of Disproportionation Of p-Methoxybenzoic-acetic Mixed Anhydride At 1COYC, Reaction Followed By Measurement Of Time (min. )Blank Sample Blank @T p-Methoxybenzoic-acetic Mixed Anhydride 20 25 35 98. 98, 98, 98, 98. 98, OT. OT. O7. 98. oT. O7 OT . oT. 97. OT. Ge. 92, aa 92. Soecific MEF HOO WOM OCHO NOR NOW NW!) el. el. el. 23. 23. 23. 25. 25. 25. eT, oT, 57. 28, 29. 28. 30. 30. 30. 31. 31. 31. ec Om ON@MOM MOU} WRAY WON SINAN NON 98. o7. 98. 98. 08. we 98, O77. 97. of. 98, ov. OT. O77. oT. oT. OT. 92, Ol. 92. OOF Woh he Uw MWUIN COOr.w} Wor @OfFUW Rate Constant ec. 22 22 a4, 24, 24, 26, 26. 26. 27. 27. ev. 29. 29 31 31 31 38 x 10° 109 2052 4.5285 086 171 171 4.1438 183 060 135 3.8328 073 826 780 3.5963 814, 686 -774 3.3668 29. .589 2503 3.1672 508 434 .602 2.8953 579 644 3 moles7+ 0.65965 0.617398 0.583518 0.555856 0.527214 0.500678 0.461695 Concentration” Ave 50 Log ave 42 (liters.mole71) 1 min. 6.3973 6.8351 7.2319 7.5918 8.003 | 8, 4286 9.1402 ~ haters -131- Rate Of Disproportionation Of p-Methoxybenzoic-acetic Mixed Anhydride At 100°C Reaction Followed By Measurement Of Time (min. )Blank Samole Blank p- Methoxybenzoic Anhydride 10 15 20 25 Sp OT. Ov. OT. OT. OT. 98. ST. oT. oT. oT. 98, 97 . O77. 97. ecific OF HOF WWM} NOAA OOO WOOO NwO ee) we) Ol. Gl. Sl. 90. GO. 90. 88. 88. 88. Mow WNW BOO Foi ~] FOO ONKF O77. O77. 98. 98, 98. 98. 98. OT. OT. OT. 98. ov. WON MOO OW” O.WW oO COMJLO Rate Constant avo nwmNon @f Ave.t2 Log ave. Lo 428 469 4428 B91 91.806 91.930 9.887 S887 S.943 .378 . 468 2365 .612 597 -597 022 920 855 - 663 O47 .678 .60 x 1073 moles7! min.” L 1.0701 1.0884 1.2441 1.1681 1,2205 0, co) 1 0290411 . 036790 .050068 058468 . 067477 . 086540 1.3044 0.115412 Concentration? (liters.moles an 8.7108 co . 9206 9.1827 XO ~3370 9.5057 9,8814 10,515 1 Liters Rate Of Disproportionation Of p-Methoxybenzoic-acetic Mixed ~132- Anhydride At 100°@ Reaction Followed By Measurement Of Time (min. )Blank Sample Blank 4T p-Methoxybenzoic Anhydride OQ An 10 20 40.5 45 OT. OT. O77. CG a ° OT. O7. OT. 97. OT. oT. OT. o7. G a e OT. Of. O77. O77. o7. O77. OT. 97. OT. 97. ST. OT. (@) me e OT. O77. Specific wwN rn y ~ OMnM WOO DAwOMO ONO ONO ON CO NOOO WDOCO DANO Ol. Ol. gl. 87. 87. 86. 84, 83. 84. 79. 79. 76. 76. 76. 73. 73. 73. veer 70. 70. 68, 68. 68. 66. 66, 66. 64, 64, 64. roo Fu NFU OW! FEU Odo OnNF Nor Wor WNW OF. oT. of. oT. oT. oT. OT. OT. oT. 97. 97. O77. Ne 98. oT. O7. ae 98. OT. 97. 97. OT. ST. ST. oT. OT. O77. O77. 98. 6 7 7 , 8 9 WOO OWMO NN@®D Won OWN OVIWO NOW Wicew Rate Constant 93 93 93 88 80 88 85 85 85 81 81 81 78 78 78 75 75 75 2 72 72 69 69 67 67 67 65 65 65 1.34 x 1073 moles7+ min. Ave. 0 Log AST 346 545 .968 - 900 . 809 B45 -787 KT .288 . 186 2394 2220 203 .318 .114 243 140 .122 .085 131 . 667 698 .703 484 .519 553 2541 573 543 1.0699 0. to ~ 1245 . 1646 22301 .2780 .4810 25255 OQ. . 106530 .123949 241984 . 156820 . 170560 . 183310 Coneentration_ (liters.moles7+) 1 -1 8. WO LO. 10. ll. 11 8183 309 729 185 .600 ~O19 Liters Rate Of Disproportionation Of Benzoic~acetic Mixed Anhydride -1 33- Time At 100°C. Reaction Followed By Measurement Of Benzoic-acetic Mixed Anhydride (min. )Blank Sample Blank %T 43. 42, He, 43, AL, 43. 45. AS, 45. 46, 46, 46, 49, 4B, 19) 10 15 20 25 30 35 90. 90. 85. 86. 86. 86. 87. 87. 87. 86. 85. 85. 87. 87. 87. 59. 89, 89. 88. 88. 88. 88. 88. 88. Specific QO OVPO =~] COLO Ono FOO WOW Ga pop OWF FWoO WI Ww LO ONO OOM FED Ow C.D CH OO MW LON © 90. 8S. 89. 86. 86. 86. 7. 87. 87. 86. 85. 85. 87. 87. MONO NOM WOWO ONC eRXe) 87.1 89. 89. 89. 88. 88. 88. 88. oT. 88. OOM Now Wwo Rate Constant 17 51 51 51 Bu, B54, 5h, 55. 56. 56. 58. 58. 58, 59. D9. 59. -791 61. 486 61 61 2 Ave .t2 Log ave 22 ( 778 AT. 47. 50. 50. 50. 418 746 546 691 634 516 ~ 340 .690 323 693 961 954 206 257 651 342 31.0 694 920 672 931 228 I 2.0987 0.321980 ~ 9753 0.295636 1.9411 0.288052 1.8295 0.262325 KH .7812 0.25071 1.7113 0.23325 6733 0.223578 je 1.6198 0.209466 x 1072 moles~+ min. Concentration liters.mole~+) 7. Ne) “10. 10, 10. 1184 ~7527 9568 7372 1420 1767 2514 O421 ~1 Liters 1 Rate Of Disoroportionation Of Benzoic~acetic Mixed Anhydride At 10C0C. Reaction oliowed By Measurement OF Benzoic-acetic Mixed Anhydride . ~ 1 . -] Time x Concentraticn ~ (min. )Blank Sample Blank @T Ave.tO Log ave =o (liters.mole7!) 9c.0 42,2 90,0 46.888 O oc.0 42.3 CO.0 47.000 2.1269 0.328260 6.9822 cO.1l 42.5 ¢cc.90 46.961 co.5 45.4 ¢cc.7 50.110 5 $0.5 45.7 90.2 50.581 1.9875 0.29831 7.6832 co.2 45.4 90.5 50.246 89.5 47.2 89.5 52.737 10 89.5 47.3 89.6 52.819 1.8048 0.277564 8.2575 80,4 47.2 89.5 52.766 89.9 50.7 GO.2 56.022 15 CO.1 50.3 $0.2 55.795 1.7871 0.252145 9.0900 $0.2 50.5 SO.0 56.048 90.9 53.2 $1.2 58.784 ; 20 91.3 53.1 Gl.1 58.2203 1.7121 0.233526 9.8148 G1.0 53.0 $1.0 58.209 90.0 54.4 80.7 60.545 25 89.6 54.5 89.5 60.859 1.6562 0,221¢0112 10.4604 CO.O 54.0 89.9 60.033 91.2 57.0 $1.3 62.465 30 $1.3 57.0 S1.2 62.500 1.6006 0.204282 LL.21°7 G1.2 56.9 01.0 62,458 ~2 -] ; 1 a oat te de a) . a . - s Specific Rate Constant = 2,82 x 10 moles min. Liters Rate Of Disproportionation Of Benzoic-acetic Mixed Anhydride At 1c0OlCC., Reaction Followed By Measurement Of penzoic-acetic Mixed Anhydride Time Slank Sample Blank aT Ave 42 Log ave.42 Concentration” © G2.0 43.5 91.7 47.359 O O15 3.2 O1.6 47.187 2.1159 0.325469 7.0414 o1.4 43,2 91.5 47.238 91.8 45.8 91.6 49.836 5 92,0 45.9 91.8 4s, ols 2,0032 0.301724 7.5663 o1,7 45.8 91.6 49.¢S72 92.0 47.A 92,0 51.521 10 61.0 U7.0 $0.6 51.790 1.9392 0.287624 7.0687 92.0 47.2 91.7 51.388 90.4 48.7 90.2 53.931 15 60,2 49.0 $0.5 54.233 1.8487 9.266868 8, 2485 GO.5 48.9 90.3 54.092 91.3 50.4 91.3 55,202 20 91.2 50.8 91.5 55.610 1.8043 0.256312 B.ohe2 91.3 0.6 91.2 55.452 91.8 53.4 92.0 58.106 a5 C2.0 53.5 $2.0 58.152 1.7205 0.235655 °.7260 $2.1 53. i, S1.7 58.106 91.2 54.8 91.2 60.087 30 91.1 54.3 92.4 59.505 1.6729 0,223448 10.2574 O1.3 54.5 91.2 59.726 92.0 57.1 92.3 61.964 35 ge2.4 57.0 92.0 61.822 1.6160 0.20844C 10.9959 92.1 57.0 92.2 61.855 1.5 60.5 91.8 66,012 6C 91.8 60.4 61.8 65.795 1.5176 0.181058 12.6589 o1.6 60.5 $1.8 65.868 Specific Rate Constant = 2.16 x 1073 moles7? min. ~ Liters ~136- Rate Of Disproportionation Of Benzoic-acetic Mixed Anhydride At 100°C, Reaction Followed By Measurement Of Benzoic-acetic Mixed Anhydride Time Blank Sample Blank eT Ave +2 Log ave.+° Concentration’ + 85.0 39.7 85.0 46.705 0 85.2 39.8 85.3 46.686 2.1422 0.33086 6.9274 85.1 39.7 85.1 46.650 83.7 41.7 83.7 49.820 5 84.0 41.7 84.0 40,642 2.0123 0.303693 7.5470 84.2 41.8 84.2 49,614 84.3 44.6 84.6 52.812 ' 10 85.0 44.8 84.9 52.736 1.8042 0.277416 8.2619 85.1 44.o B49 52.823 85.6 46.2 85.5 54.000 15 83.5 45.6 83.5 54.610 1.8362 0.263918 8.6945 83.5 45.7 83.4 54.763 85.7 48.5 85.7 56.592 20 85.6 48.4 85.8 56.476 1.7696 0.247874 9.2466 85.6 48.3 85.5 56.458 85.6 50.2 85.6 58.644 25 85.8 50.4 85.9 58.707 1.7044 0.231570 ~ 9,8976 85.8 50.3 85.7 58.658 86.0 52.1 86.2 60.511 oo . 30 86.0 52.3 86.1 60.778 1.6460 0.216430 10,5900 85.9 52.4 86.0 60.965 83.0 53.2 83.2 65,222 AG 83.3 53.3 83.4 65.146 1.5331 0.185569 12.3512 83.4 53.4 83.2 65.306 90.8 60.0 90.5 67.217 ; 120 92.0 60.1 92.4 67.353 1.4867 0.172203 13.3098 92.3 60.0 92.2 67.208 Specific Rate Constant = 2.44 x 1073 moles~* min. | haters ~127- Effect Of Added Suostances On The Rave Of Disprcportionavion Of Benzoic-acetic Mixed Anhydride. Reaction Folicwed Ry Measurement Of Benzoic-acetic Mixed Anhydride At 1OO”“C Substance Concentration, Time +, Concen- Added Moles Liters ~min. Biank Sample Blank Ave tration” gO.1 42.8 90,2 Benzoic O.20 0 90.7 43.1 90.8 2.1049 7.0697 Acid 92.0 43.3 $1.1 9.8 45.7 89.7 " 0.20 5 89.6 45.6 86.9 1.565C 7.8129 90.0 45.8 89,8 . 38°,2 48.6 89.4 2 i" 0.20 10 89.6 48.7 89.6 1.8362 8.6614 89.3 48.6 89.5 90.0 51.3 89.9 | " 0.20 15 90.0 51.2 90.01.7555 9.3784 89.8 51.1 86.6 89.7 53.0 89.6 © " 0.20 20 SO0.0 53.2 89.9 1.6896 10.063 89.7 53.2 86.6 7 W NM ro}! oO Pa ead © vias , -3 ey -l .. Specific Rate Constant = 3 “ain. moles Liters 90,2 42.9 90.) sodium O.i gms G YO.7 43.2 00.7 2.1025 7.1001 Acetate Suspension 90.5 42.1 90.8 90.4 47.0 90.5 " " 5 90.6 AZ. 90.5 1 $367 7.9844 90.8 47.2 91.0 C0.5 50.2 90.4 " " 10 90.4 50.1 ¢o.4 1.8031 O.°522 SoO.4 50.1 90.3 $0.6 52.7 So.7 " " 15 SO.& 52.8 So.g 1.7206 3.7246 S1.0 2.9 Si.l G1.2 56.0 91.3 " " 20 O1.4 56.1 91.5 1.6297 10.8067 S1.7 56.3 91.8 Specific Rate Constant = 3.64 moles~+ min.7~1 Liters 1 ~138- Substance Concentration Time To Concen- Added Moles Liters min. Blank Sample Blank AVG o> tration” S2.1 43.6 S1.8 Powdered Suspension QO S1l.4 43,3 01.7 2.1154 7.0437 Glass C.10 2m CG1.4 43.1 G1.6 82.7 41.8 83.¢ ' " 5 BL 41.6 84,0 2.0119 7.54ch 84.3 A1,9 el e 87.1 us.1 87.4 " i" 10 87.7 44.9 87.6 1.9403 7.5619 87.3 LS.1 87.3 85.4 46.3 85.3 " " 15 83.6 45.7 83.6 1.8422 8, 6379 83.6 45.7 83.7 87.8 49.2 88.0 | " i 20 87.2 48.8 87.4 1.789 C.0653 87.5 48.7 87.3 : ~3 a Specific Rate Constant = 2,010 x 10°” moles min, ~ Livers 85.2 30.7 85.1 O 85.3 20.5 85.3 2.1434 6.9223 ~~ 85.2 39.6 85.0 85.2 42,8 85.0 _ 5 85.3 42,8 85.5 1.9965 7.5603 ~~ _ 85.6 42,9 85.7 B44 Abbey B4.8. __ 1c «85.3. 44.5 85.2 1.9065 = 8.1788 oo 7 85.2 44.5 85,0 86.3 H6.4 86.5 _ 15 86.8 46.6 87.0 1.8627 8.4844, _ 87.2 46.S 87.4 85.8 48.7 85.0 20 85.9 48.6 85.5 1.7627 9.3103 —_ | 85.7 48.7 85.6 1 1 Specific Rate Constant = 2.44 x 1073 *»oles* min.” Liters ~13¢- substance Concentration Time -. Concen- | Added Moles Liters ~ min. Blank Sample Blank Ave =~ tration™+ ce. 42.6 1.7 Acetic 0.2 GO 01.6 4UB.1 91.5 2.1117 7.0601 Acid G14 42,2 62.5 93.2 4UT7.6 S8.2 " Q.2 5 €3.3 7.7 G3.4 1.9570 7.8722 O3.4 47.8 93.7 93.9 5l.4 93.7 " 0.2 10. «CALL 52.6 G4.2 1.8265 8.7607 GC4.O 8652.5 Sh 4 23.6 53.3 93.9 © " 0.2 15 ¢3,8 52.3 63.5 1.7590 9g. 3448 63.5 53.2 03.7 93.6 55.8 93.4 — " 0.2 20 93.5 55.8 63.4 1.6757 10.223 03.3 55.7 93.5 Specific Rate Constant = 3.0666 x 1073 moles? min.7+ Liters 26.0 48.7 90.3 Acetic 0.3 QO CO.L 42.7 90.2 2.1096 7.0031 Acid 60.2 42.8 90.2 89.7 45.9 89.5 " 0.3 5 86.6 46.c 89.8 1.9503 7.8994 89.8 46.1 89.9 86.9 49.3 90.2. " 0.3 10 89.8 4o.4 90.0 1.8216 8.8002 o0.1 49.6 CC.3 $0.8 52.1 9C.7 ; " 0.3 25 C0.6 52.6 90.8 1.7426 O.5107 SO.2 51.7 89.9 $1.2 54ULS =Sl.1 " 0.3 20 91.0 54.7 90.8 1.6591 10.424 91.3 55.0 1.5 -2 _ — Specific Rate Constant = 3.33 x 10° moles 1 min. I Liters -140- Summary Of Data For N-Acyl Bara-Toluidine Systems Principal Wave lengths 1. N-(acetyl)-p-toluidine --- 7.269 microns 2, N-(benzoyl)-p-toluidine --- 6.676 microns 3. N-(p-methoxybenzoyl)-p-toluidine --- 8.534 microns 7 N-(p-nitrobenzoyl)-p-toluidine --- 7.393 microns Beer's Law Curves 1. N-(acetyl)-p-toluidine a. At N-acetyl wave length Log ae) = 1,271 (N-acetyl) + 0.000 b. At N-benzoyl wave length Log - = 1.211 (N-acetyl) + 0.00025 c. At N-(p-methoxybenzoyl) wave length Log ae = 0.0858 (N-acetyl) + 0.000 d. At N-(p-nitrobenzoyl) wave length Log ze = 0.3150 (N-acetyl) + 0.000 N-(benzoyl)-p-toluidine MN a. At N-acetyl wave length Log #2 = 0.196 (N-benzoy1) + 0.000 b. At N-benzoyl wave length Log x = 1,814 (N-benzoyl) + 0.008 3. N-(p-methoxybenzoy1 )-p-toluidine a. At N-acetyl wave length Log fe = 1,184(N-p-Methoxybenzoyi) + 0.000 b. At N-(p-methoxybenzoyl)-p-toluidine wave length Log ie = 10.810 (N-p-methoxybenzoyl) + 0.000 -141- 4, N-(p-nitrobenzoyl) p-toluidine a. At N-acetyl wave length Log ie = 0.7442 (N-p-nitrobenzoyl) + 0.000 b. At N-p-nitrobenzoyl p-toluidine Log R= 6.560 (N-p-nitrobenzoyl) + 0.000 Simultaneous Equations Derived From Above Data A. For the benzoic-acetic mixed anhydride at 7.26S microns; 0.196 (benzoyl) + 1.271 (acetyl) = Log a at 6.676 microns; 1.814 (venzoyl) + 1.211 (acetyl) = Log ae - 0.0083 B. For p-methoxybenzoic-acetic mixed anhydride at 7.269 microns; 1.184 (methoxybenzoyl) + 1.271 (acetyl) = Io Log - at 8.534 microns; 10.81 (methoxybenzoyl) + 0.0858 (acetyl) = For p-nitrobenzoic-acetic mixed anhydride at 7.269 microns; 0.7442 (nitropdenzoyl) + 1.271 (acetyl) = Q2 Log Tr at 7.393 microns; 6.565 (nitrobenzoyl) + 0.3150 (acetyl) = qu Io Og + ~142- Beer's Law Curve For N-Benzoyl p-toluidine At N-Acetyl- p-toluidine Wave Length 7.269 Microns In Chloroform Concentration Blank Sample Blank &4T Ave Ae Log a 98.2 90.1 98.1 91.798 0.1908 98.0 90.0 98.2 91.743 1.0895 0.03720 98.3 90.2 98.2 91.806 98.5 91.8 98.4 93,245 0.1526 98.3 91.7 98.2 93.333 1.0712 0.029870 98.2 91.7 98.1 93.428 © 98.5 93.5 98.4 94.972 O.1145 98.2 93.5 98.4 95.116 1.0524 0.022184 98.3 93.4 98.4 94.966 98.5 95.2 98.5 96.6465 0.07632 98.5 95.3 98.4 96.800 1.0337 0.014394 98.4 95.2 98.4 96.747 Log => = 0.1960 (N-Benzoyl tolu) + 0.000 ~143- Beer's Law Curve For N-Benzoyl-p-toluidine At Its Peak Concentration Blank Sample Blank 6.676 Microns In Chloroform 0.1908 - 0.1526 21145 OQ oO 7632 25724 2) co) .3816 io) . 1908 98. 98. 98. UI OVO WW NOTORS) “U1 OF MR) MH MN U1 O* = 1.814 (Benzoyl tolu) 43, 43, 43, 51. 51. 51. 60. 60. 60. 67. 67. 76. 76. 75. 82. 82. 82. 89. 89. 89. UI -u WOAO ™~ ON CO OV I= OrRO “SN ON NOVO 98. 98. 98. An Nolven = UU WO NO Ono Oru UI OVI ra SO. SO. Gl 2501 ~399 478 . 987 .063 O14 .288 452 .367 »,860 -726 .865 ~275 ~235 094 .628 612 ~{13 826 965 .112 To Ave == 2 + a 2402 9219 .6294 4323 -2953 21954 0992 0.008 Log a2 0.352028 0.283728 0.212028 0.156033 0.112372 0.077514 0.041078 -144- Beer's Law Curve For N-Acetyl-p-toluidine At N-(p-Nitro- penzoyl)-p-toluidine Wave Length 7.393 Microns Concentration Blank Sample Blank 4T Ave. #0 Loga2 97.8 84.0 97.8 85.8859 0.2121 97.8 84.1 97.6 86.079 1.1623 9.065328 07.7 84.2 97.8 86.138 . ' 07.7 86.4 97.8 88.388 0.1697 97.8 86.3 97.7 88.286 1.1320 90.05385C 07.8 86.4 97.8 88.343 97.7 90.1 97.6 92.268 0.1273 97.6 90.0 97.5 92.260 1.0834 0.034790 97.5 90.0 97.4 92.355 97.7 92.2 97.6 94.418 0.08495 97.5 92.1 97.6 94.413 1.0588 0.024818 97.7 92.2 97.7 94.370 Log 2 = 0.3150 (Acetyl tolu) + 0.000 Beer's Law Curve For N-(acetyl)-p-toluidine At N-(Benzoyl)- p-toluldine Peak 6.6/7 Microns In Chloroform Lo Concentration Blank Sample Blank &T Ave. 40 Log a 98.2 64.2 98.1 65-410 C.2176 98.1 63.8 S8.1 65.035 1.5330. 0.18554 98.2 64.0 S8.0 65.239 . 98.2 70.5 98.2 71.792 0.17408 98.3 70.3 98.0 71.625 1.3944 0.144384 98.2 70.4 998.1 71.726 98.2 76.0 98.0 77.471 0.13056 98.0 76.4 98.1 77.919 1.2872 0.109648 98.1 76.2 S8.1 77.675 98.0 83.0 S8.1 84.650 0.08704 98.2 82.9 S8.2 84.419 1.1826 0.072836 08.0 83.0 S8.2 84.607 98.2 89.9 98.4 91.408 0.04352 98.3 85.8 98.2 91.399 1.0939 0.038980 98.5 90.0 98.4 91.416 98.3 98.9 98.2 95.572 0.02176 98.0 93.7 98.1 95.563 1.0469 9.019908 98.2 93.8 98.4 95.422 Log 2 1.211 (Acetyl tolu) + 0.00025 i -145- Beer's Law Curve For N-A.cetyl-p-toluidine At Its Wave Length 7.209 Microns In Chloroform Concentration moles Liters! Blank Sample Blank 0.2677 0.2176 0.1741 0.1306 0.0870 0.0435 0.02176 OF O77. 56 MOO 6 8 EMH KHON Hee OOrwno ~N OWO AW, Ay, Ay 535 53. 53. 59. 59. 59. 67. 67. 67. 76. 76. 76. 86. 86. 86. 3 F 6 WN PW NNO HMO HOO re 92.2 9e. 92. MFO 97. 98, 27 O77. OT. o7. 5 O 25 HHO wWwo Ww Po HHO OrO ONO es 45.412 45.658 45.720 54.054 54.026 54.156 60.296 60.377 60.244 68.874 68.804 68.734 TT 896 77.794 TT 936 88.163 88.220 88.028 O4.177 94,069 94.171 Ave, £2 T . -) ~1931 4640 » 2847, ~ 1345 0622 1.271 (Acetyl tolu) + 0.C0O Log Io —T 0 0 - 341060 .266964. ~ 219823 ~ 16554 . 108603 . 050860 . 026202 -146- Beer's Law Curve For N-Acetyl-p-toluidine At (p-Methoxy- benzoyl )~p-toluidine Wave Length 3.534 Microns Ir: Concentration Chloroform moles liters’ Blank Sample Blank 95.4 90.5 95.4 0.262 95.8 90.8 95.6 95.2 90.1 95.0 96.4 92.3 96.3 0.2096 96.2 92.2 96.1 96.1 92.3 96.3 95.8 92.7 96.0 0.1834 95.9 92.6 95.8 95.7 92.7 95.9 96.2 93.9 96.3 0.1332 96.1 93.6 96.0 96.4 94.0 96.3 Log . 0.0858 (Acetyl tolu) + 0.000 a O4. 94, 94, 95 95 95. 96. 96. 96. 97 863 879 THe 746 .B91 O45 663 609 764 2558 97. O77. iio 560 Ave 0 1.0545 1.0431 © 1.0343 1.0254 Her 0.023045 0.018322 0.014646 0,.010892 ~1L47- Beer's Law Curve For N-(p-methoxybenzoyl )-p-toluidine At N-acetyi-p-toluidine {.209 Microns In Chloroform Concentration Blank Sample Blank 4%T Ave, 32 Logi? 97.1 80.2 97.0 82.637 0.0692 96.8 79.9 96.6 82.626 1.2101 0.082825 96.7 80.0 96.9 82.644 Oo 96.8 83.5 97.0 86.171 0.0554 97.2 83.8 97.4 86.125 1.1610 0.06483 97.4 83.9 97.5 86.095 96.9 86.5 97.1 89.129 0.0415 97.0 86.6 97.2 89.186 1.1216. 0.049828 96.9 86.3 96.7 89.152 95.6 88.6 95.7 92.629 0.0277 95.8 88.7 95.7 92.637 1.0793 0.03314 95.6 88.5 95.4 92.670 95.8 92.2 95.7 96.292 0.0138 95.8 92.3 95.9 96.296 1.0386 0.016452 96.0 92.5 96.2 96.253 Log 42 = 1.184 (p-MeO tolu) + 0.000 Beerts Law Curve For N-(p-Methoxybenzoy1)-p-toluidine At Its Wave Length 6.534 Microns © Concentration Blank Sample Blank 4T Ave. ae “Lo <2 97.5 46.8 97.6 47.975 0.02995 97.7 46.7 97.6 47.823 2.0881 0.319752 97.6 46.6 97.5 47.770 98.4 55.2 98.5 56.069 0.02395 98.3 55.1 98.4 56.024 1.7837 0.251318 98.4 55.2 98.4 56.097 98.4 63.2 98.4 64,227 0.01796 98.3 63.2 98.2 64.325 1.5543 0.191628 98.4 63.1 98.4 64.126 98.4 73.1 98.4 74.288 0.01198 98.4 73.2 98.4 74.390 1.3452 0.128786 98.3 73.1 98.4 74.326 98.2 85.4 98.3 86.921 0.00598 98.3 85.3 98.4 86.731 1.1524 0.061502 98.3 85.2 98.3 86.673 Log 42 10.81 (pMeOtolu) + 0.000 -148- Beer's Law Curve For N-(p-Nitrobenzoyl)-p-toluidine At Concentrati moles Liters~+Blank Sample Blank N-Acetyl-p-toluidine Wave Length on 7.209 Microns In Chioroform 0.0940 0.0752 0.0564 0.0376 0.01880 Log 96. 96. 96. 95. 96. 6. 95. o5. 95. 96. 96. 96. 95. 95. 95. Lo —e I Beer's Law Curve Fro Who OV OCOVK1’O MW PP CON \O 82. 82, 81. 84, 84, 84, 86. 86. 86. 90. 90. 90. 92, 92. 92. COO \O AFF WOW UIN\O IW NNO FMR NOOO NOW) RM EO a 977 150 »135 TAL .870 .850 662 -709 .052 811 .818 . 873 .922 B14 .706 Ave. 19 L 1.1757 1.1386 1,19032 © 1.0657 1.0329 = 0.7442 (p-Nitro tél) + 0.000 Log *? 0.07030 0.056368 0.042658 ' 0.027596 0.014058 For N(p-Nitrobenzoyl)-p-toluidine At Its Wave Lengtn 7.393 Microns In Chloroform Concentration Blank Sample Blank %T 58. 58. 58. 64. 64, 64, 7O. 10, 70. 79. 87. 87. 87. 0.03484 0.02788 0.02093 0.01394 0.006974 Log OT. OT . OT. oT. oT. oT. OT. OT. O7 . O7. OF . 97 . ST. oT. 97. Io L NOT WANA WOO ONC Moco WOOO WIFE COno FOO ANN OT. Te 98. OT. OT. 97. 97 OT. OT. oT. OT . O7. ST. oT. of. ANN DANA WAN NANO oOWoOW} 60. 59. 60, 65. 65. 65. .606 643 (17 80. 80. Te 72 (2 81 091 959 061 848 916 984 ohe ge . 086 89. 85. 89. = 6.560 (p-Nitro tolu) + 0.000 810 856 754 Ave, Lo r 1.6656" 1.5170 1.3776 1.2347 1.1134 0.221666 0.18099 0.139116 0.091565 0.046655 Reaction Medium _ Wave Length Blank Sample Blank -149- Cleavage Of Benzoic-acetic Mixed Anhydride With p-Toluidine Benzene 50 w/w Acetone- Benzene 93.4 26.2 93.5 28, 7.269 93.5 26.3 93.6 28. 93.6 26.4 93.4 28, 93.3 26.4 93.2 28, 6.676 93.3 26.5 93.3 28. 93.2 26.3 93.1 28. Melting point of mixture = 149- van Ave 22 Logie 036 113 3.5558. 0.550936 220 289 . 403 3.5324 0.548068 234 150°C Concentration N-acetyl = 0.431956 Concentration N-benzoyl = 0.0098277 Acetylation ratio = 44.0 93.5 50.0 93.5 53.475 7.269 93.2 49.8 93.3 53.404 1.8722 0.272356 93.1 49.7 93.2 53.354 93.7 50.2 94.0 53.489. 6.676 O4.0 50.3 94.1 53.482 1.8718 0.272262 93.8 50.1 S4.0 53.354 Melting point of mixture = 148.6-150°C Concentration N-acetyl = 0.213414 Concentration N-benzoyl = 0.005665 Acetylation ratio = 37.7 Reaction -150- Medium Wave Length Blank Sample Blank eT Ave => Log -F Acetone 50 w/w Acetone- Water 36.4 SAA 38.538 GA.5 7.269 94.0 36.2 94.2 38.469 2.5964 0.414368 94.1 36.3 94.3 38.535 95.3 36.1 95.2 37.900 6.676 95.1 36.1 95.0 37.980 2.6358 0.420916 95.0 36.0 94.8 37.934 Melting point of mixture = 148.4-150°c Concentration N-acetyl = 0.32425 Concentration N-benzoyl = 0.0109437 Acetylation ratio = 29.6 S4.5 29.9 94.7 31.606 7.269 G4.6 29.8 94.3 31.551 3.1606 0.499768 94.5 30.0 94.7 31.712 94.3 29.5 94.2 31.299 6.676 94.1 29.4 94,2 31.226 3.1949 0.504456 94.3 29.6 94.4 31.372 Melting point of mixture = 149-150°C Concentration N-acetyl = 0.49747 Concentration N-benzoyl = 0.0117124 Acetylation ratio = 42,5 -151- Cleavage Of p-Methoxybenzoic-acetic Mixed Anhydride With p-Toluidine Reaction Medium Wave Length Blank Sample Blank 4T Ave £9 Log “42 97.8 64.9 97.9 66.326 Bezene 7.269 97.9 64.8 98.1 66.088 1.5095 0.178835 98.C 65.0 98.0 66.326 97.8 92.0 97.8 94.069 8.534 97.8 91.9 97.7 94.015 1.0634 0.026694 97.8 92.0 97.9 94.021 Melting point of mixture = 143°c-144° Concentration N-acetyl = 0.139427 Concentration N-p-methoxybenzoyl = 0.00136202 Acetylation ratio = 102 50 w/w 97.0 45.6 97.2 46.961 Acetone- 7.269 97.1 45.7 97.3 47.016 2.1297 C.328320 97.2 45.5 96.9 46.883 96.8 88.0 97.0 90.815. 8.534 97.1 88.3 97.3 90.843 1.1009 0.041750 97.2 88.2 97.0 90.834 143 .5°c=144.6°C Melting point of mixture Concentration N-acetyl = 0.256616 Concentration N-p-miethoxybenzoyl = 0.00182417 Acetylation ratio = 140 Reaction -152- Medium _ Wave Length Blank Sample Blank %T Ave 5° Log 42 98.9 54.6 98.7 55.263 Acetone 7.269 98.6 54.5 98.6 55.273 1.8089 0.257396 98.9 54.7 98.9 55.308 98.9 Gl.1 98.8 92,159 8.534 98.9 91.2 98.9 92.214 1.0846 0.035270 98.7 91.1 98.9 92.206 Melting point of mixture = 143°c-144°c Concentration N-acetyl = 0.200965 Concentration N-p-nethoxybenzoyl = 0.0016676 Acetylation ratio = 120 50 w/w 97.6 35.7 97.9 36.521 Acetone- 7.269 97.9 35.8 98.0 36.549 2.7379 0.437414 Water 08.0 35.8 98.1 36.511 98.2 83.8 98.1 85.379 8.534 98.3 83.8 98.1 85.492 1.1709 0.068523 08.5 84.0 98.4 85.322 Melting point of mixture = 144° C=145°C Concentration N-acetyl = 0.340763 Concentration N-p-methoxybenzoyl = 0.00363418 Acetylation ratio = 93.7 ~153- Cleavage Of p-Nitrobenzoic-acetic Mixed Anhydride With p-toluidine Reaction Io Io Medium Wave Length Blank Sample Blank 4T Ave.=- Log 61.6 95.6 64.536 61.7 95.4 64.607 1.5487 0.189966 61.5 95.2 64.566 Benzene 7.269 LOO KO UVOVOI A Oyu 95.8 44,1 96.9 46.009 7.393 95.9 44.2 96.0 46.065 2.1724 0.336940 96.1 44.2 96.0 46.017 Melting point of mixture = 125-131°C Concentration N-acetyl = 0.12286 Concentration N-p-mitrobenzoyl = 0.045428 Acetylation ratio = 2.70 50 w/w 094.8 47.0 95.0 49.525 Acetone- 7.269 94.9 47.0 94.8 49.551 2.0212 0.305612 94.7 46.8 94.6 49,445 94.5 40.5 94.4 42,879 7.394 94.6 40.5 94.6 42,811 2.3470 0.37051 94.6 4o.4 94.5 42.728 Melting point of mixture = 126-132°C Concentration N-acetyl = 0.213477 Concentration N-p-nitrobenzoyl = 0.046194 Acetylation ratio = 4.66 ~154- Reaction r Io Medium Wave Length Blank Sample Blank GT Ave <2 Log > 39.4 93.: 93.4 3 42,206 Acetone 7.269 93.1 39.2 93.0 42.127 2.3718 0.375020 92.9 39.2 93.1 42.150 93.2 26.2 93.1 28.126 7.394 93.0 26.2 93.4 28.111 3.5644 0.551988 93.5 26.1 93.4 27.929 Melting point of mixture = 126.5-131°C Concentration N-acetyl = 0.25308 Concentration N-p-nitrobenzoyl = C.071937 Acetylation ratio = 3.52 50 w/w 92.8 48.0 93.0 51.668 Acetone - 7.269 93.0 48.1 93.1 51.692 1.9341 0, 286482 93.1 48.2 93,2 51.744 92.9 33.5 93.0 36.040 7.394 93.1 33.5 93.2 35.963 2.7768 0.443548 93.2 33.6 93.3 36.032 Melting point of mixture = 124,.8-133.1°C Concentration N-acetyl = 0.19121 Concentration N-p-witrobenzoyl = 0.058387 Acetylation ratio = 3.28 Apparent First Order Rate Constants For The Hydrolysis of Para Substituted Benzoic-acetic Mixed Anhydrides p-Methoxybenzoic-acetic Mixed Anhydride Xe = 9,630 milliliters t (min) Keo -Xy t (min) Xoo -Xp 19.0 5.09 234.5 3.40 39.2 4.59 255.0 3.25 59.0 4.06 295.0 3.08 79.0 4.03 324.0 2.95 99.0 4.10 360.0 2.89 119.0 3.97 395.0 2.72 159.0 3.83 455.0 3.09. 179.0 3.83 485.0 2.40 202.5 3.56 515.0 2.21 Corrected first order rate constant = 1.41 x 107° Sec.7! p-Nitrobenzoic-acetic Mixed Anhydride Xe = 22,35 Milliliters ¢ (min) Xea -Xt t (min) Keo - Xt 1 10.77 15 9.37 ed 10.35 18 9.15. 3 10.33 el G.00 4 10.25 24 8,82 6 9.93 28 8.77 8 9.83 32 8.26 10 9.73 37 8.04 Le 9.53 43 7.74 51 7.33> Corrected first order rate constant = 23.7 x 107° Sec. 7+ -156- Benzoic-acetic Mixed Anhydride xe = 14.885 milliliters t(min) Keo - Xz t (min) Keo - Xp 1.2 7.311 32.0 6.525 4.0 7.204 36.0 6.406 8.5 7.085 39.0 6.335 12.5 6.985 43.0 6.262 16.0 6.898 47.5 6.205 20.0 6.845 54.0 5.035 24.0 6.715 60.0 5.935 28.0 6.655 70,0 5.765 : 80.0 5.527 Corrected first order rate constant = 3.77 x 1079 Sec. 74 PART II. STUDIES OF THE ALPHA-CHYMOTRYPSIN CATALYZED HYDROLYSIS OF SPECIFIC SUBSTRATES THE KINETICS OF THE Q& CHYMOTRYPSIN CATALYZED HYDROLYSIS OF THREE N-ACYL-~L-TYROSIN HY DRAZIBES It has oceen known for a long time that the enzyme Achymotrypsin can catalyze the hydrolysis of certain acylated and unacylated amino acid esters, amides and hydrazides. The work which is reported here is concerned with the kinetics of the hydrolysis of three N-acyl-L-tyrosinhydra- zides; the N-trimethylacetyl, the N-dichloroacetyl and the N-formyl-L-tyrosinhydrazides. The results are shown in Table 1. Table 1 The Kinetic Constants for Three N-Acyl-L-tyrosinhydrazides k3(min.~1)x1073 K,x1073 N-Formy1-L-tyrosinhydrazide 0.068 12.8 N-Trimethylacety1-L-tyrosinhydrazide 0.30 29.2 N-Dichloroacetyl-L-tyrosinhydrazide 0.53 3,84 At the time that this work was undertaken tt was be- lieved that the course of the hydrolysis of the N-acyl-L- tyrosinhydrazides could be predicted upon the basis of the -158- classical Michaelis-Menten formulation. However, it has subsequently been shown by other investigators in these laboratories that this belief may not be valid. The values of the various kinetic constants which are reported here are calculated on the basis of the classical mechanism and must be regarded as provisional. Therefore no attempt has been made to correlate any of the results either among themselves or with respect to data determined by other in- vestigators. The classical approach, which was found to be valid for the hydrolysis of the amides, assumes that the enzyme and substrate react reversibly with one another to form an enzyme-substrate complex. This complex may then undergo a transformation resulting in the formation of the free enzyme and the products of hydrolysis. Ks E+S — LES] Ae 4Pji4P, Koz LEMS! | ALPY_ & [es] Ces J at On the basis of this reaction mechanism it is possible to derive the following relationship: It Where [S] Substrate concentration at time t [E] = Total enzyme concentration [ES]= Enzyme-substrate complex concentration V Velocity at time t iH} t By taking the reciprocal of the above equation, the foilowing equation is obtained: yo Ks+ist ly = ; (Ys) + Re [EMS Raa When. t=0, thn S=5, and V=Vo 7 eB Equation C establishes a relationship which enables one to estimate the kinetic constants K, and Ka from a knowledge of the initial velocities at various substrate concentra- tions. A complicating factor arises when the products of -160- hydrolysis combine with the enzyme and hence prevent its further interaction with the substrate. The effect of product inhibition may be included in the basic formulation by defining the following equilidrium: Ke E+Pp==(|FP] Ke= LEICPI [EP] This results in the modification of equation B to inelude K_: inelude p zt K So-S \ __ = Pa (15) * ste + He s ) D It is possible to neglect the effect of product inhibi- tion if the experiment is designed so that only a small percentage of hydrolysis is followed and if it is assumed that Ke> Kp. This is the basis upon which the experiments which led to the results shown in Tables 3, 5 and 7 were carried out. It is predicated upon the validity of equation B. By integration of equation B' it is possible to derive equation D. RE= Ms (it %,) a t S25 (I ky )—D -161- From this relationship it is seen that a plot of in “ofS versus “928 should be a straight line whose slope is a function of S., K,, and K,. For the hydrolysis of p? several N-acyl-L-tyrosinamides this linear result has been obtained. W. Lands of these laboratories has recently observed the hydrolysis of N-acetyl-L-tyrosinhydrazide over a rela- tively large extent of reaction. When the data for this reaction were examined according to equation D a decided deviation from linearity was obtained. The data indicate that the interaction of the enzyme and product may be greater than any that has been observed so far, with the apparent Ky much greater than Kye The data for the hydrolysis of N-formyl-L-tyrosinhydra- zide and N-trimethylacetyl-L-tyrosinhydrazides (Tables 3 and 5) do not show any influence of product interaction. This is presumably due to the small extent of reaction which was observed and the tendency of any random error in the data to be magnified when plotted according to equation D. N-Dichloroacetyl-L-tyrosinhydrazide hydrolyzes at a somewhat more rapid rate. When the data for its hydrolysis were examined by equation D a classical K, of approximatdy . p 3.8 x 107 was indicated (Figure 8). The effect of this apparent classical product inhibition was corrected for by application of the Lands modification of the Jennings correction (recorded as f, in Table 7). -162- N-Trimethylacetyl-L-tyrosinhydrazide was found to be an extremely soluble substrate. It was possible to work with solutions which were as concentrated as 0.24F. This substrate concentration was much higher than had ever been obtained before. The Ammonium Salt Of N-Acetyl-L-Cysteic Acid Carboxyl- amide As A Substrate During his work on the structure of insulin, Sanger observed that &chymotrypsin catalyzed the hydrolysis of a peptide bond which involved the carboxyl group of cysveic (1) acid. This was the first time it was suggested that @ chymotrypsin would act upon cysteic acid peptides. It was hoped that this would prove to be a general ob- servation and that simple cysteic acid derivatives would serve as suitable substrates. N-Acetyl-L-cysteic acid carboxylamide (I) was synthesized to serve as a model sub- strate. ae & NH SF cheat — (2) N fz0 CHs It was found, however, that it was not possible to demonstrate (Table 8) that any appreciable hydrolysis had ~163- occurred when chymotrypsin was allowed to act upon (I). In like manner it was not possible to demonstrate that (I) has any appreciable ability to act as a competitive inhi- bitor (Table 9). Under these conditions the ammonium salt of N-acetyl-L-cysteic carboxylamide has no ability to in- fluence the behavior of Xchymotrypsin, There is an accumulation of evidence to indicate that a negative charge is associated with the active site of % chymotrypsin. In view of this evidence it is not sur- prising that the negatively charged cysteyl ion fails to interact with the enzyme. The cysteic acid peptide link in insulin which was hydrolyzed is a bond in which the amino group of serine is (2) involved. It is suggested that at some time, perhaps during the preliminary treatment of the insulin molecule with performic acid, an "N" to "0" shift had occurred, in this process the carboxyl group of the cysteic acid residue had migrated to the adjacent hydroxyl group converting the peptide link into an ester bond. It is well known that a chymotrypsin will catalyze the hydrolysis of a wide variety of esters, As a corollary to this work an experiment was run to determine the effect of sulfate ion on the activity of achymotrypsin. The results are shown in Table 10. It is evident that under these conditions sulfate ion does not affect the enzyme. -164- The Synthesis Of The N-Acyl-L-tyrosinhydrazides The synthetic route to N-trimethylacetyl-L and N- dichloroacetyl-L-tyrosinhydrazides is outlined below. SelsoH HOD CHecH # Oz Hs (L) Nisco KHCQ, Ree HoGargits +, HeOH ——=> « | | HO-C>-CHe CH C-NH-NH, <——__ HoS>-che-en—L “OCR Hs on wa dag (2) R R L-Tyrosine Ethyl Ester (1) One mole of L-tyrosine (Merck & Co.) was suspended in 2s liters of absolute ethano1 {3) contained in a five liter ‘three neck round bottom flask. The center neck contained a mechanically sealed electrically driven stirrer. One of the side necks contained a drying tube filled with calcium chloride. Into the remaining neck was inserted a delivery tube for the admission of anhydrous hydrogen chloride. The whole assembly was packed in ice and maintained at o°c. The cold ethanolic suspension was saturated with an-— hydrous hydrogen chloride, the process taking 24 hours. A -165- clear solution was obtained and allowed to stand over night at room temperature. The amber colored ethanolic solution was then evapo- rated to dryness under reduced pressure (25 mm.) on a steam bath. The crude L-tyrosine ethyl ester hydrochloride was ground to a fine powder and suspended, with the aid of a mechanical stirrer, in one liter of dry chloroform cooled to O°C. Two hundred ml. of a cold saturated solution of ammonia in chloroform was added over a period of 30 minutes. . After an additional 4 hour the precipitated ammonium chlor- ide was filtered off and the chloroform evaporated to dryness under reduced pressure. The crude L-tyrosine ethyl ester was recrystallized twice from dry ethyl acetate. Final M.P = 108,5-109°C [ a = + 20.5 (methanol) Yield = 69% The material was kept in the dark and stored at 5°C until used. Alkyl Acid Chlorides Trimethyl, dichloro and trichloroacetyl chlorides were synthesized by the method of H. C. Brown, (4) This method is -166- based upon the reaction of the desired acid with benzoyl chloride. The method is applicable only to acid chlorides which have a boiling point lower than benzoyl chloride, . qs haat n 9 -(2 + RC- —— R-c-ce + -OH @n ce C OW JD tiee qr BaP. Yield Trimethylacetyl Chloride 104-105°C 85% Dichloroacetyl Chloride 106-107.5°C = 744% Trichloroacetyl Chloride 116-118°¢C 53% Acylation of L-Tyrosine Ethyl Ester Five one-hundredths mole of L-tyrosine ethyl ester was dissolved in 300 ml. of dry ethyl acetate contained in one liter Erlenmeyer flask. The solution was cooled to -5 C in an ice salt bath. The cold solution was agitated with an emulsifying, vibrating, electrically driven stirrer. Twenty-five thousandths mole of the acid chloride was then added. After two minutes 50 ml. of 0.05 N potassium car- bonate solution was added, two minutes later another 0.025 mole portion of the acid chloride was added followed two minutes later by another 50 ml, portion of potassium carbon- ate. The process was repeated until four samples of acid chloride and the potassium carbonate solution had been added. During the process of addition the entire system was completely emulsified. -~167- The cold solution was allowed to separate into two phases. The ethyl acetate phase was washed with 6 N HCl and then twice with cold distilled water, The ethyl acetate solution was dried by being shaken with calcium chloride and then with anhydrous potassium carbonate. The ethyl acetate was removed by evaporation under re- ‘duced pressure. The product that was obtained was not treated further but converted directly to the hydrazide. Crude Yield Physical State N-Trimethylacety1l-L-Tyrosine Ethyl Ester 55% oil N-Dichloroacetyl-L-Tyrosine oo Ethyl Ester 48% gummy solid ~The Conversion of the N-Acyl-L-tyrosine Ethyt Esters to the Corresponding Hydrazides. The crude esters were dissolved in 23 times their weight of anhydrous methanol. The solutions were heated to boiling and a 50% excess of hydrazine hydrate was quickly added. ‘The cool mixtures were allowed to stand over night. The volumes of the methanolic solutions were reduced and the hydrazides allowed to crystallize. The yield of the crude materials averaged 80% The compounds were recrystallized from methanol-water mixtures. N-Dichloroacetyl1-L- N-Trimethylacety1l-L-tyrosinhydrazide tyrosinhydrazide M.P. = 179-180° c “MP. = 193-193.5° [ a hd = 29,0°(water) [a nd = 412,5° (40% ethanol) ~168- Elementary Analysis Elementary Analysis Cale = 60.19 Cale =z 43,20 10} Cc Cale = 7.53 Cale = 4,25 H H Found = 7.52 Found = 4,24 Cale = 15.05 Cale # 13.70 N N Found = 15.12 Found = 13.75 Overall yield from Calc = 23.23 tyrosine ethyl ester = 18% Cl Found = 23.31. Overall yield from tyrosine ethyl ester = 13% N-Formy1-L-tyrosinhydrazide The synthesis of N-formyl-L-tyrosinhydrazide was simi- lar to the above sequence except that the acylation was. carried out in a different manner, Five one-hundredths mole of L-tyrosine ethyl ester was Gissolved in 250 ml. of 80% formic acid and placed in a 500 ml. three neck, round bottom flask equipped with a mechanically sealed electrically driven stirrer. The flask was protected from the atmosphere by a calcium chloride drying tube. 43 ml. (0.5 mole) of acetic anhydride was Slowly added to the agitated mixture in the flask. The rate of addition was such that the temperature did not rise above TO°C. One half hour was required for the addition. ~169- The mixture was allowed to stand over night at room temperature and then concentrated to a syruv under vacuum. The syrup was dissolved in 200 mls. of ethyl acetate and thoroughly washed with 3 N sodium hydroxide solution, then with 6 N HCl and finally with distilled water. The ethyl acetate was removed by evaporation under reduced oressure. The syrup obtained (60% yield) was then treated with hydrazine in the manner indicated above. N-Formy1-L-tyrosinhydrazide M.P. = 218°C (Decomp.) [ x 78 = + 21.4 (30% ethanol) Elementary Analysis Cale = 53.75 Cale: = 5.95 C H Found = 53.70 © Found = 6.0} Cale = 18.85 N Found 18.80 Over all yield from L-Tyrosine Ethyl Ester = 15% -170- The Synthesis of the Ammonium Salt of N-Acetyl-L- cysseic Acid Carboxylamide The synthetic route used to obtain this compound is outlined below. O a NH » Sto { NH, ce —C > ? HcQ- S © I S -CHy CH-C-0 9 “CHS cH -C-ocHs Nis NH, cQ Secu eue w Br, DCH CH-C-o¢ i slgen <., ep (2 (rr) ~CH3- cH d-0c H 3 HN-E Ae Nis + ©) i Nig O-S—cHy- cy NH. & HN GT CHS Gy) -171- L-Cystine Dimethyl Ester Dihydrochloride (I) This compound was synthesized from commercially avail- able L-cystine in a manner exactly analogous to the synthe- sis of L-tyrosine ethyl ester (p. 164). The product was recrystallized from methanol. Yield 89% N,Nt Diacetyl-L-cystine Dimethyl Ester (II) The free base, cystine dimethyl ester, is an unstable syrupy substance, which slowly decomposes to free sulfur, alamine and other substances. (>? Therefore it is neces- sary to carry out the acetylation of this compound without first preparing the free base. | | One tenth mole of the finely powdered diester dihydro- chloride was suspended in two liters of dry tetranydrofuran contained in a five liter three neck round bottom flask protected from the atmosphere by a drying tube filled with calcium chloride. The suspension was cooled to -5°C. One mole of dry redistilled triethylamine was added. The sus- pension was maintained for one half hour. Twenty-two hundredths mole of freshly redistilled acetyl chloride was Slowly added. The mixture was allowed to stir for four hours. The triethylamine hydrochloride was removed by filtra- tion and the solvent removed by distillation under reduced pressure. The yield of the crude crystalline material was 71%. N-Acetyl-L-cysteic Acid Carboxylmethyl Ester (III) Five-hundredths mole of the diacetyl dimethyl cystine derivative was placed in a 500 ml. three neck flask, and 250 ml, of distilled water containing 60 ml. of concentrated hydrochloric acid was added. The mixture was slowly stirred with a mechanical agitator. After solution was complete, liquid bromine was slowly added. The addition was con- tinued until the bromine color was no longer discharged. (about 0.25 mole required.) The resulting solution was evaporated under reduced pressure. The product crystallized from the residue in a 75% yield, M.P. = 186 (With Decomposition. ) The Ammonium Salt of N-Acetyl-L-cysteic Acid Carboxylamide (IV) Four hundredths mole of the crude N-acetyl methyl ester derivative was dissolved in one liter of dry methanol con~ tained in a two liter single neck round bottom blask and cooled to O°C, The solution was saturated with anhydrous ammonia and while still at O°C the glass stopper was wired in place. The flask was placed in a heavy walled creck and allowed to stand at room temperature for five. days. The solvent was stripped off at reduced pressure and the product recrystallized from anhydrous ethanol, M.P. = 201-202°C pop = 9,279 (water) Cale = 26.39 Cale = 5.74 Elementary Composition C H Found = 26.35 Found = 5.77 Cale = 18.50 Cale =14.10 N Ss Found = 18.50 Found =13.96 Overall yield from Amino Acid = 18% Analytical Procedure In order to get the information necessary to calculate k., and Ke the following general procedure was adopted. 3 A buffer solution was made up by dissolving enough tris hydroxymethylaminomethane (Tham) to make a O0,.2F solu- tion. Concentrated hydrochloric acid was added drop wise to the solution, which was maintained at 25°C until the desired pH was approached. The pH of the stock buffer solution was read on a Beckman pH meter. The stock buffer solution was then diluted ten fold and its pH observed. Final adjustments were made to the stock solution until the diluted solution had the desired pH value. All the runs were carried out at & buffer concentration oF 0,02 F (except where noted) and at a pH of 7.8. The pH optimum curves (Figures 1, 3 and 5) of the three substrates ~174- Seem to indicate a pH optimum which is slightly lower than this. However, since the previous work was done at 7.8 it was decided to carry out these investigations at this value also, The reaction between hydrazine and p-dimethylamino ‘benzaldehyde is the basis of the colorimetric procedure (6) used to follow the course of the hydrolysis. HN-NH, -- 2 3g o & > To start the procedure of a typical run, eight ml. of 4 the substrate solution, equilibrated to 25°C were pipetted “nto a 10 ml. volumetric flask. One ml. of the stock 25°C ouffer solution was then added. At zero time, one mi. of 25°C enzyme solution was pipetted into the volumetric flask, the pipette allowed to drain for ten seconds, the flask stoppered and inverted 12 times and placed in a 25° + 0.02°¢ constant temperature hath. At various times aliquot sanples were olpetted into a 10 ml. volumetric flask which contained one ml. of 1.60 F HCl and one mil. of a 10% solution of p-dimethylamino benzalde- hyde in 100% ethanol. The solution was then diluted to the calibration mark and the optical density determined at us WI millimicrons with a Beckman model D spectrophotometer. The color samples were allowed to stand about 15 minutes , at room temperature before the optical density was deter- mb mined. Subsequently it was found that it was shat the azine solution should be incubated at 25°C before the optical density was determined. At the conclusion of each run the pH was checked to be certain that it had remained constant. The enzyme concentration was one milligram of protein per ml. of solution. The enzyme sample analyzed 14.444 protein nitrogen. i Often it was noted that the enzyme solutions were quite ‘turbid, which was presumably due to the precipitation of the enzyme from the acidic solution. This causes the plot of optical density versus time to intersect the optical density axis rather than pass through the origin (Figure 6). This necessitates that a blank correction be applied to each optical density reading. This blank correction must be obtained for each run by extrapolation of the optical density versus time curve until it intersects the optical hed a 4 aensi x ow ci GS axis. Subsequently it was found that the turbidity could be greatly reduced by adding seven ml. of water to the 10 ml. volumetric flask which contained the standard -~176- acidified p-dimethylaminobenzaldehyde solution. In this way the enzyme solution was pipetted into a solution whose initial acid concentration was lower and precipitation did not occur. The observed optical densities were corrected by sub- traction of the plank values and recorded in Tables 3 to 10 as D,(corr.). From these values the first order term was calculated. V, was estimated both from a first order plot and a zero order plot. The average of the two values was used in the calculation of Ke and Ke. The final results were obtained by a "least squares" calculation on the data as expressed by equation B. _— hr ~~ ~~ we) —_ -177- REFERENCES 2 F, Sanger, E, 0. P. Thompson - Biochem. J. 53 — 353 (1953) D. F. Elliott - Biochem. J. 50 542 (1952) -L. F. FPieser - Experiments in Organic Chemistry - page 358 (1941) D. C. Heath and Company H. C. Brown - J.A.C.S. 60 1325 (1938) R. D. Coghill - J. Biol. Chem. 114 A1g-Hed (1936) R. Lutwack - Ph.D. Dissertation, California institute of Technology (1954) The Relative Rates Of The & Chymotrypsin Catalyzed Hydrolysis Of N-Trimethylacetyl-L-tyrosinhydra- zide At Different pH Values Time of Hydrolysis = 122 min. So = 2.56 x 1073 molar Initial pH Observed Optical Density Final oH 6.25 0.706 -6,23 6.65 1.142 6.63 7.13 1.396 7.12 7.30 1.475 7625 7.40 1.495 7.37 7.75 1.519 7.7e 8.14 1.352 8,12 8.30 1.350 8.27 8.42 1,226 | 8.37 7.36 1.472 7.33 7.50 1.505 7.48 7.63 1.534 7.60 7.65 1.521 7.62 7.71 1.491 7.69 7.84 1.480 7.83. 8.00 1.478 7.96 O.D. 1.7 1.6-—- 1.4}— 1,2+~— 1,1j;— 1,0-— -179- Figure 1 pH Optimum Curve for Trimethy lacetyl-L-tyrosinhydrazide Optical Density vs. pH to 7.5. 8.0 8. An pH Table 3 The & Chymotrypsin Catalyzed Hydrolysis Of N-Trimethylacetyl-L-tyrosinhydrazicde Initial Substrate Concentration = 0.240 Molar Extent of Hydrolysis = 0.48% Blank = 1,65 Time D,(corr)* 1n (Dee -D,) In De (Min. ) . ¥ (De —D,) 2 0.33 6.81872 0.00203 Mh 0.89 6.81811 0.00264 6 1.19 6.81778 0.00297 8 1.65 6.81729 ~ 0.00346 10 2.17 6.81672 0.00403 12 2.55 6.81620 0.00455 14 2.95 6.81577 0.00408 16 3.31 6.81535 0.00540 Vo = 5.68 x 107? moles.min. 7+ Tnitial Substrate Concentration = 0.20 Molar Extent of Hydrolysis = 0.22% Blank = 0.830 Time D,.(corr)” In(Dee -D,) in Deo (min. ) v (DS De) 2 0.194 6.63724 0.00133 A O. 4d 6.63691 0.00166 6 0.860 6.63662 0.00195 8 1.036 6.63612 0.00245 10 1.016 6.63615 0.00242 12 1.322 6.63575 0.00285 14 1.532 6.63547 - 0.00310 16 1.706 6.63524 0.00330 Vo = 4.34 x 1072 moles*min. 7+ * 3 4 = % 4 4- 4 ™ Wey j " This column was obtained by subtraction of the blank value from the original data Table 3 Continued . . 4 Initial Substrate Concentration = 1.21 x 10 ~ Molar Extent of Hydrolysis = 1.19% Blank = 1.35 me % in Dew (ite D.. (corr) In(Dep -D,) Deo -D;) 4 0.73 6.120962 0.00234 8 1.39 6.12818 0.00378 Le ei 6.12661 0.00535 16° 2.69 6.12534 0.00062 20 3.41 6.12376 0.008206 au 4.09 6.12259 0.009037 28 4.85 6.12068 0.01128 32 5.53 6.11911 0.01285 Vo = 4.28 x 1072 moles+min7+ Initial Substrate Concentration = 3.02 x 107+ Molar Extent of Hydrolysis = 1.27% Blank = 0.360 Time x in | Deo (min. ) D,(corr ) In ( Deo -D.) (Doo =D: ) 2 0.105 A745 0.00403 Ab 0.270 4.74261 0.00537 6 0.365 4.74091 0.00717 8 0.565 4.73916 0.00982 10 0.768 4.73739 0.01169 12 0.933 4.73673 -0,01225 14 1.113 73489 0.01319 16 1.253 473401 0.01407 18 1.470 4.73209 0.01599 5 Vo = 2.18 x 10 ~ moles-min.7~ *% This column was obtained by subtraction of the blank value from the original data Table 3 Continued 4 4 ct 4 + 2 -2 “Initial Substrate Concentration = 1.048 x 107° Extent of Hydrolysis = 2.10% Blank = 0.033 Time 1 cr). | ~ (iin. ) D, (corr) in(Dee D,) (Deo Dy) Qt 0.027 3.68541 0.00421 dy 0.217 3.68345 0, 00617 6 0.310 3.68083 0.00879 8. O.414 3.67819 0.01143 10 0.514, 3.67567 0.01405 12 0.610 3.67304 0.01658 - iA 0.742 3.66996 0.01966 16 0.840 3.66751 0.02211 Vo = 1.38 x 1075 moles+min.~* Initial Substrate Concentration = 7.56 x 1073 Extent of Hydrolysis = 2.70% Blank = 0.195 (nine) D, (corr) In(Deo -D,) Deo Dy, i 0.103 5.65533 0.01057. 8 0,248 5.65124 0.01446 12 0.412 5 6446 0.02124 16 0.490 5.64134 0.02436 20 . 0.654 5.63586 0.02984 ah 0.761 5.63203 0.03367 28 0,885 5.62758 0,03812 32 1.006 5.62322 0.04248 36 1,107 5.61957 0.04613 Vy = 8.33 x 107° moles+min. 7? * . 4 * 2 * This column was obtained by subtraction of the blank value from the original data Table 3 Continued Tnitial Substrate Concentration = 7.49 x 19073 Extent of Hydrolysis = 3.80% Blank = 0.114 ant * (min. ) D, (corr) In(De ~-D,) Deo -D, 3 0.061 3.34686 0.00614 6 0.163 3.343 61 0.00939 | 9 0.283 3.33005 0.01395 12 0.488 3.33535 0.01763 17 0.581 3.32848 0.02452 20 0.683 - 3, 32e472 0.02828 Ol 0.836 3.31921 0. 03379 28 0.996 2.31339 O. 03961 V.= 9.76 x 107° moles‘min.7+ ie) Initial Substrate Concentration - 5.99 x 107? Extent of Hydrolysis = 3.07% Blank = 0.172 | In Deo (nie \ D,(corr)” 1n (Deo -D,) (Deo =D, ) min.) . 2 0.030 3.12047 0, 00848 4 0.098 3.11745 0.01200 6 0.150 3.11514 O,01431 i) Q,211 3.1122 0.01703 10 Q.276 3.10954 0.01991 12 0.309 3.10807 O. 02138 14 0.404 3.08823 0.02557 16 0.426 3.07921 0.02668 . -6 Vo = 7.41 x 10 moles-:min.~+ % . : . . mo This column was obtained by subtraction of the blank value from the original data Table 3 Continued AD Initial Substrate Concentration = 4.49 x 10 ~- Extent of Hydrolysis = 1.87% Biank = 0.150 (min. ) D, (corr) in(De -D,) Deo =D.) A 0.088 2,82821 0.01389 8 0.185 2.82233 0.02077 | 14 0.340 2.81294 0.02916 16 0.393 2.80973 0.03237 20 0.476 2.80569 0.03641 au. 0.560 2.79954 0.04256 28 0.653 2.79396 0, 04814 32 0.749 2.78815 0.05395 Vv fe) = 6,23 x 107° moles-min.”~ ‘Initial Substrate Concentration = 2.99 x 1079 Extent of Hydrolysis = 4.35% Blank = 0.152 . , in Deo fae Di (corr)” 1n (Deo -D,) (Dee = Dy 10 0.018 1.70992 0.03379 15 0.154 1.70339 0.04032 20 0.203 1.69442 0.04929 25 0.256 1.68649 0.05722 30 0.272 1.68074 0.06297 35 0.365 1.66421 0.07950 “HO 0.398 1.65794 0.08577 V. = 2.38 x 107° moles+min. ~~ O * This column was ootained by subtraction of the blank value from the original data Table 3 Continued Initial Substrate Concentration = 2.99 x 107? Extent of Hydrolysis = 4.35% Blank 0.152 re , * in Deo time D, (corr) in(De -D,) (De -D.) (min. ) _2neene t v dy 0.043 2 41859 0.0169 8 O.114 2.41235 0.02315 10 O.144 2.40971 0.02576 15 0,247 2.40078 0.03472 20 0.316 2.35330 0.05220 Ou 0.372 2.38006 0.05544 2 0.439 2 37434 0.06126 32 0.512 2.36812 0.6723 Vy = 4,12 x 107° moles+min. ~~ Tnitial Substrate Concentration = 1.08 x 1073 Extent of Hydrolysis = 7.20% Blank = 0.067 Time se In Deo (min. ) D, (corr) In(De -D,) Tes =D.) 4 0.038 1.39158 0.02568 8 0.063 1.38604 0.03117 12 0.084 1.38003 0.03718 16 0.113 1.37270 0.04451 20 0,152 1.36533 0.05188 Ou 0.159 1.36098 0.05623 28 0.174 1.35196 0.06525 32 0.204 1.34937 0.06784 Vi = 1.59 x 107° moles:min. 72 x This column was obtained by subtraction of the blank value from the original data 4K) © o be o) Od ?) ie} aT) ci ead 3 Cc i) a Initial Substrate Concentration = 7.4¢ x 1074 e Blank = 0.107 ~*~ in Doo mis Fd 7 & ~ (min. ) _ Di (corr) In(Dog -D,) (Deo -D.) 5 0.023 1.00404 0.04654. 10 0.040 0.99780 0.5278 . 15 0.066 0.98816 0.06242 20 0.081 0.98257 0.06801 25 0.103 0.97340 0.07628 30 0,125 0.96595 0, 08463 35 0.145 0.95831 0.09227 LO 0.168 0.94045 0.10113 -~6 . -1 Vo = 1.12 x 10 moles*min. Initial Substrate Concentration = 6.59 x 1074 Extent of Hydrolysis = 6.15% Blank = 0.09 Time ; * In ___Doo | (nin. ) D, (corr) in(Dee -D,) TDoo -D,) 5 0.021 0.87705 0.04562 10 0.041 0.86919 0.05348 15 0.060 0.86120 0.06147 20 0.074 0.85527 0.06740 25 0.096 0.84587 0.07680 30 0.115 0.83768 0. 08h0¢ 35 0.136 0.82855 0.09412 ho 0.154 0.82066 0.10201 Vo = 1.02 x 197° moles-min.7+ “this column was obtained by subtraction of the blank value from the original data wnt od Table 3 Continued ~ 8 oe 4 , . - —eh Initial Substrate Concentration = 5.76 x 10 } Excent of Hydr Tin * hs (nin. ) D, (corr) in(Qbe -D,) 5 0.003 0.74953 10 0.020 0.74146 15 0.038 0.73237 20 0.057 0.72368 25 O.O77 0.71393 30 0,004 0.70557 35 O.111 0.69350 WO. 0.125 0.69014 "7 -1 Vy = 8.50 x 107! moles+min, ~~ 0.03963 0.04770 - 0.05679 0.06548 0.07523 0.08359 0.09566 0.09902 Initial Substrate Concentration = 4.91 x 1074 Extent of Hydrolysis = 8.55% Blank = 0.083 Time * (min. ) Di(corr) — In(e -D,) hh 0.012 0.57672 le 0,035 0.56588 20 0.052 0.55446 28 0.078 0.54582 36 0.097 0.52886 Ad O,119 0.51581 52 O.14e2 6.50197 60 0.162 0.48980 Vo= 7.27 x 107 moles+«min.71 Oo 0.05285 0.06374 0.07521 0.08385 0.10081 0.11386 0.12768 0.13987 *This column was obtained by Subtraction of the blank value from the original data : initial Substrate Concentration ~ Looe nat Ce Table 3 Continued Extent of Hydrolysis Blank = Time (Min. ) 6 12 18 au 30 36 Ap 18 54. Vv 0,050 e) % Dd, (corr) 7.12 x 107! moles+min 0.011 0.030 0.070 0,067 0.081 0.100 0.114 0.124 0,152 8.10% =H Oh x 107 In Deo. In(Dee -D,) Dao —D,. u 0.60213 0.03391 0.59167 0, 04437 0.57987 0.05517 0.57098 0.06406 0.56531 0.06973 oO. 55158 0, 08446 0.53606 0.09098 0.52176 0.097S7 0.5406 0.11328 -1 This column was obtained by subtraction of from the original data the blank value -189- FIGURE 2 foo {SO Zoo 250 300 350 Yor jt Jt. ¥50 i ---t-- = ot v7 “Se { §se 6 Oe web Wo 135 — {3c 123 -—- 20 "WS ; 1D fas i i i eee een oe FIGURE 2 plot oF ¥ vs. Ve FOR THE Ne 50 HYDROLYSIS OF TRIMETHYLAGETYL~L— TYROSINHYORZIDE Bco Bse Goo gso jo~ jose \ {it { ; ‘ : yoo 1180 {402 2250 1300 isso 2700 Lp Ete bs theo Zh 2608 26 Fi 76 ee dete “4850 2 Leg {Sos Ey Roos Rael ey 5 Table 4 The Relative Rates Of The & Chymotryosin Catalyzed Hydrolysis Of N-Formyl-L-tyrosinnydrazide At Different pH Values Time of Hydrolysif = 135 min. [So ] = 1.2 x 107* molar Tnitial pH . Observed Optical Density Final pH 6.20 . 0.252 6.20 6.70 0.318 6.65 7.00 0.382 6.92 7.25 0.411 7.21 7.50 0.420 TAT 7.80 0,422 7.78 8.25 0.360 8.27 8.42 0,324 BAS 8.35 0.365 8.30 8.63 0.322 8.54 7.80 0.390 7.79 7.88 0.420 7.85 7.78 0.434 7.77 7.75 0.434 7.72 7.70 0.438 7.66 7.65 0.395 7.64 7.60 0.439 7.62 7.54 0.429 7.51 7.58 0.436 7.55 ~191- Figure 3 pH Optimum Curve for N-Formy1-L-tyrosinhydrazide Optical Density vs. pH O.D. 0.46 — O44 LL o.42 . 0.40 — 0.38 — . 0,36 — 0.34 0,32 1 . 0.30 0.28 /- 6.26 }— 0,24 -—- 0,22 — 6.0 6.5 7.0 7. 1 pH Table 5 The % Chymotrypsin Catalyzed Hydrolysis Of N-Formy1-L-tyrosinhydrazide Initial Substrate Concentration = 13.47 x 107° Molar Extent of Hydrolysis = 1.23% Blank = 0,220 ‘Time | Dp, (corr) In(De -D D ae (Min, ) Ath - ° A 0.040 6.23830 0.00501 8 0.112 6.23690 0.00641 12 0.194 6.23529 0.00802 16 0.270 6.23380 0.00957 20 0.334 6.23255 0.01076 Ou 0.406 6.23113 0.01218 28 0.497 6.22933 0.01398 32 0.561 6.22808 0.01523 36 0.622 6.22687 0.01644 = 49.40 x 107! moles-min.-1 Initial Substrate Concentration = 11,23 x 1072 Molar Extent of Hydrolysis = 1.37% Blank = 0,210 : in Deo (itin.) . Dy (corr) Ln (Dee -D,) (Deo -D,) 4 0.029 6.05547 | 0.00559 8 0.106 6.05367 0.00741 12 0.174 6.05216 0.00890 16 0.241 6.05049 0.01057 20 0.308 6.04890 0.01216 oy 0.375 6.04732 0.01374 28 0.439 6.04581 0.01525 32 0.530 6.04365 O.OL7AL 26 0.586 6.04232 0.01874 Ve = 45.50 x 107% moles«min. ~ “This column was obtained by subtraction of the blank value from the original data Table 5 Continued initial Substrate Concentration = 8.08 x 10 molar Extent of Hydrolysis = 1.46% Blank = 0,200 In Dee (ate) Dy(eorr) — In(Deo ~D,) (Deo BEY A 0.030 3.52862 0.00668 8 0.085 3.52701 0.00829 . 12 O.141 3.52536 0.009094 16 0.216 352316 0.01215 20 278 3.52132 0.01398 alt 0.324 3.51906 0.01534 28 0.375 3.51845 6101685 32 0.440 3.51652 0.01878 36 0.502 3.51468 0.02062 Vo = 38,00 x 107 moles+min. 7+ Tnitial Substrate Concentration = 8.00 x 1079 Molar Extent of Hydrolysis = 1.52% Blank = 0,155 (min.) Yi (COPTr ; D\ Veo ear (Deo ete hy 0.026 3.41375 0.00581 8 0.075 3.41214 0.00742 12 O.143 3.40939 0.00967 16 0.195 3.40517 0.01139 20 0.247 3.40695 0.01311 ey 0.312 3.40151 0.01526 28 0.371 3.40131 0.01725 32 0,427 3.40054 0.01902 = 37.82 x 107 moles-min.7+ Xin a bey ; Lt ; . This column was obtained by subtraction of the blank value from the original data Table 5 Continued Initial Substrate Concentration = 6.74 x 1073 Molar Extent of Hydrolysis = 1.66% Blank = 0.173 Time in Doo (Min. ) D, (corr) In(Deo -D,) (Doo -D,) 4 0.027 3.23985 0.00780 8 0.077 3.23789 0.00976 . 12 0.126 3.23557 0.01208 16 0.178 3.22392 0.01373 20 0,227 3.23175 * 0.01590 au 0.275 3.22965 0.01796 28 0.337 3.22771 0.01994 32 0.386 3.22577 0.02188 364 0.428 3.22410 0.02355 Initial Substrate Concentration = 5.56 x 1073 Molar Extent of Hydrolysis = 1.72% Vo = 30.05 x 10- 7 ~ ool moles-min. . Blank = . ln . Deo _ (nis D,(corr)” In(Dee -D,) (Dee -Dr) dy 0.068 3.05055 0.00886 8 0.093 3.04037 0.01003 12 0.142 3.04704 0.01236 16 0.189 3.04481 0.01459 20 0.231 3.04280 0.01660 2, 0.279 3.04051 0.01889 28 0.317 3.03869 0.02071 2 0.370 3.03572 0.02368 Xen: . 5 : , This column was obtained by subtraction of the blank value from the original data Table 5 Continued Initial Substrate Concentration = 4.63 x 1073 Molar Extent of Hydrolysis = 1.9% Blank = O.111 Time \* in Dee wee y “why 4 or -—cacememenemnenin tau al Min.) D, (corr) In(Dee ~D,) (Deo =D.) 4 0.048 2.86340 0.00982 8 0.074 2.86271 0.01051 12. 0.136 . 2.85916 0.01406 16 0.170 285721 0.01601 20 0.203 2.85532 0.01795 - ol 0,249 2 85233 0.02089 28 0.269 2.85152 0.02175 32 0.326 2 84820 0.02505 Vy = 26.78 x 10°! moles-min.~1 Initial Substrate Concentration = 3.90 x 1072 Molar Extent of Hydrolysis = 2.66% Blank = 0.150 . eo In Dee Time . .\* ~ (Min. ) D, (corr ) in(Dee -D,) (Dee “Di hh 0,040 2. 68847 0.01276 8. 0.077 2.68590 0.01533 12 0.110 2.68233 0,01840 16 0.159 2.68115 0.02005 20 0.192 2.67856 0.02267 ek 0.236 2.67509 0.02614 28 0.265 2.67297 0.02826 30 0.308 2.67000 0.03123 36 0.372 2.66556 0.03567 V, = 23.3 x 107 moles-min.7+ % * 4 * * This column was obtained by subtraction of from the original data the odlank value Taple 5 Continued ty 4} 2 74 2 ong Initial Substrate Concentration = 3.71 x 10° Molar Extent of Hydrolysis = 2.37% Blank = 0.120 Time \* In Deo (Min. ) D, (corr ) In(Dee -D Deo -Dy) 4, 0,048 2.63813 0.001193 8 0.066 2.63684 0.01322 . 12 0.112 2.63354 0.01652 16 0.148 2.63095 0.01901 20 0.185 2.62828 0.02178 24 0,218 2.62590 0.02416 28 0,242 2.6241 0.02590 30 0.279 2.62147 0.02859 38 0.348 2.61645 0.03351 Vy = 23.55 x 10°! moles-min. + Initial Substrate Concentration = 2.08 x 1073 Molar Extent of Hydrolysis = 2.434 Blank = 0.130 Time D * In Deo __ | (Min.) De (corr) In(Dee -D,) (Deo -D;) Mt 0.029 2.05386 0.02019 8 0.038 2.05271 0.02134 12 0.062 2.04953 0, 02414 16 0.086 2, 04653 0.02752 20 0.104 2, O40 0.02965 eu. 0.127 2.04122 0.03283 28 0.153 2.03780 0.03625 32 0.171 2.03680 0.03725 36 0.192 2.03275 0.04130 Vv 40 x 10 moles-min.” * This column was obtained by subtraction of the blank value from the original data Table 5 Continued Initial Substrate Concentration = 0.926 x 1073 Molar Extent of Hydrolysis = 3.27% Blank = 0.110 Time ; * ; in Deo (Min. ) Dz (corr) In(Deo -D,) (Deo -D,.) 5 0.017 1.22730 0.03655 10 0,015 1.22788 0.03597. ~ 15 0,027 1, 22434 0.03948 20 0,021 1.22515 0.03870 a5 0.060 1.21461 0.04925 30 0.046 1.21877 0.04508 LO O.0S0 1,20567 0.05718 AS 0,100 1.20267 0.06118 50 0.113 1.19876 0.06509 V. = 6.55 x 107! moles-min. ~~ * This column was obtained by subtraction of the blank value fo) from the original data c-OTXQT " ST vas Et 58- ~ Le ct CT OL ) pep pe Z STsATo & ZL a) Gq 7 € Z aptzeapAYyUuT s0i£4-7-TAWAOY-N JO fo) apsy aug soz g *sa °A/%g Jo 4014 4 SInsty -1°o. Table 6 The Relative Rates Of The & Chymotrypsin Catalyzed Hydrolysis Of N-Dichloroacetyl-L-tyrosinhydrazide At Different pH Values Time of Hydrolysis = 40 min. {s,] = 6 x 10-4 molar Initial pH Ooserved Optical Density Final pH 6.20 0.370 6.20 6.70 0.571 - 6.68 7.09 0.702 6.93 7.25 0.852 7.10 7.80 0.869 7.78 8.32 0.685 8.35 8.25 ©.75C 8.25 8.43 0.660 8.40 8.00 0.833 7.94 7.88 0.883 7.85 7.78 0.887 7.75 7.75 0.895 7.72 7.61 0,894 7.66 7.65 0.905 7.64 7.60 0.909 7.62 7.54 0.907 7.51 7.40 0.892 7.40 7.30 0.875 7.26 ~200- Figure 5 ph Optimum Curve for Dichloroacetyl-L-tyrosinhydrazide O.D. Optical Density vs. pH 95 675 -201- Table 7 The Kinetics of the AChymotrypsin Catalyzed Hydrolysis of N-Dichloroacetyl-L-tyros inhydrazide Initial Substrate Concentration = 3.82 x 1073 Molar Extent of Hydrolysis = 9.96% Blank «= 0,150 Deo * In t D, (corr) Deo -Dt fo 2 0.298 0.01952 0.9893 3 0.458 0.03113 0.9854 4 0.580 0.4227 0.9790 5 0.758 0.05285 0.9739 6 0.905 0.06344 0.9689 7 1.032 0.07401 0.9637 9 1.338 0,09482 0.9542 10 1.454 0.10499 0.9492 V, = 4.20 x 107? moles+min.~? Initial Substrate Concentration = 3.26 x 1073 Molar Extent of Hydrolysis = 11.52% Blank = 0.120 Deo % in t D, (corr) Deo -D, fo 2 0.298 0.02562 0.9879 3 0.440 0.03748 0.9865 5 0.730 0.06216 0.9753 6 0.872 0.07448 0.9644 T 1.039 0.08915 0.9576 8 1,170 0.10083 0.9522 9 1.300 0.11254 0.9468 10 1.435 0.12584 0.9411 = 4.04 x 107> moles-min.~? *The data in this column were obtained by subtraction of the blank value from the original data -202- Table 7 Continued Initial Substrate Concentration — 3.234 x 1073 Molar Extent of Hydrolysis = 10.80% Blank = 0.125 Deo . . * t D, (corr) — ings =D, fo 2 0.275 0.02400 0.9886 3° 0.421 0.03410 0.9833 4 0.545 0.04566 0.9773 5 0.708 0.05728 0.9718 6 0.819 0.06960 - 0.9659 7 0.933 0.08013 0.9607 8 1,103 0.09163 0.9554 9 1.197 0.10303 0.9502 10 1.345 0.11422 0.9448 Vo = 3.84 x 107 moles *min. ~~ Initial Substrate Concentration = 2,855 x 1073 Molar Extent of Hydrolysis = 12.24% Blank = 130 D * Ing 8 t _(corr) -D, fo 2 0.268 0,03874 0.9876 3 0.398 0.05112 0.9815 4 0.510 0.06812 0.9762 5 0.602 0.07038 0.9701 6 0.770 0.08621 0.9661 7 0.902 0.10949 0.9579 8 1/4 1.055 0.11544 0.9508 9 1/4 1.192 0.12976 0.9396 10 1/2 1.339 0.14522 0.9374 1 V. = 3.47 x 107? moles *min.~ o *The data in this column were obtained by subtraction of the plank value from the original data -203- Table 7 Continued Initial Substrate Concentration = 2.45 x 107 Molar Extent of Hydrolysis = 11.13% Blank = 0.100 *% t Dy (corr) Ing -Dy fo 3 0.362 0.04000 0.9801 - yo 0.471 0.05098 0.9747 5 0.574 0.07341 0.9640 6 0.730 ~ 0.08600 0.9588 7 0.830 0.09577 0.9536 8 0.940 0.10802 0.9478 9 1.062 0.11790 0.9433 Vo = 3.39 x 10-5 moles emin, 72 Initial Substrate Concentration = 2.31 x 1073 Molar Extent of Hydrolysis = 11.314 Blank =- 0.130 D co *% t D, (corr) Inns -Dy fy 2 0.229 0.02351 0.9882 3 0.286 0.03531 0.9827 4 0.396 0.04711 0.9765 5 0.515 0.05868 0.9711 6 0.608 0.07078 0.9602 7 0,702 0.08295 0.9595 8 0.805 0.09514 0.9538 9 0.925 0.10761 0.9480 10 0.971 0.12000 0.9423 Vo = 2.86 x 107? moles «min. ~~ *The data in this column were obtained by subtraction of the blank value from the original data aed Table 7 Continued . oe Initial Substrate Concentration = 1.85 x 10°” Molar Extent of Hydrolysis = 12.54% Blank = 0,062 x in De ; t D, (corr) Deo -D; fy 2 0.183 0,025134 0.9810. 3 0.288 0.03922 0.9808 He 0.345 0.05309 0.9737 5 0.469 0.06697 0.9672 6 0.603 0.08068 0.9605 7 0.624 0.09409 0.9543 8 0.719 0.10760 0.9480 Q 0.787 0.12087 0.9419 10 0.888 0.13402 0.9359 _ ~1 Vo = 2.54 x 10 2 moles-+min. Initial Substrate Concentration = 1.50 x 1073 Molar Extent. of Hydrolysis = 12.16% Blank = 0.130 % in Deo & | Dd, ( corr) | Deo ~D.. fo 2 0.160 0.05187 0.9861 2 0.220 0.06285 0.9809 4 0.280 0.07040 0.9851 5 0.360 0.08931 0.9682 6 0.435 0.10176 0.9614 ve 0.502 0.11675 0.9558 8 0.565 O.14717 0.9489 g 0.608 0.15674 0.9459 10 0.697 0.17372 0.9379 5 Vg = 2.23 x 10 ? moles:min. 7 mae 4 *e This column was obtained by subtraction of the blank value from the original data -205~ Table 7 Continued Initial Substrate Concentration = 9.24 x 107" Molar Extent of Hydrolysis - 12.97% Blank = 0,095 t D, (corr) ings ~Dy fo 20 0.073 0,04876 9.9892 3 0.175 0.07041 0.9792 4 0.198 0.08665 0.9715 5 0.214 0,09472 0.9689 6 0.327 0.12732 0.9530 7 0,348 0.13410 0.9499 8 O.409 0.15406 0.9395 9 0.458 0.17039 0.9337 . wn 5 - V5 = 1.45 x 1075 moles-min,~+ Initial Substrate Concentration - 9.09 x 1074 Molar — Extent of Hydrolysis = 15.016% Blank = 0,100 Dee t D, (corr) * i Deo =D, fo 2 0,134 0.07611 6.9831 3 O.14s 0.08044 0.9783 4 0.202 0.09733 0.9704 5 9.259 0.11548 0.9620 6 0.318 0.13461 9.9558 7 0,256 0.14713 0.9477 & 0.405 0.16351 0.9405 9 0.456 0.18085 0.9327 10 0,523 0.23982 0.9228 1 Vo = 1.54 x 1079 moles-¢min. *The data in this column were obtained by subtraction of the lank value from the original data -206- Table 7 Continued Initial Substrate Concentration =.= 3.02 x i074 Molar Extent of Hydrolysis = 16,034 Blank = 0,085 * in_Deo ‘ D, (corr) Deo -D, £6 2 0.039 0.10369 0.9831 3 0.071 0.13989 0.9689. 4 0,076 0.14991 0.9666 5 0,095 0.16891 0.9581 6 0.115 0.18991 0.9489 7 0.143 0.21991 0.9367 8 0.149 0.22491 0.9339 9 0.167 0.24591 0.9257 10 0.185 0.26691 0.9129 Vo = 5.83 xX 107 moles«min,7? *the data in this column were obtained by subtraction of the blank valve from the original data b OM | roy a a T *uTuW” soTOW apt zerpAYU TT sorh4-J-TAZaOVOUOTYUOTG-N G JO sTsATOIpAH 94 JO YOTg dapdg ouag QQ eaInsta "2. 9°O GO GEO 8 T | T i} 7 “ e re) _Ot ¥ OO*t = (paqodais00) A oO ¢-Ol x Ocg°e = § l 8) OT (up) 4 g~d od UI ytro fT°0 eT°O Tito OT*O 60 gord 40°O 90° GUrD TOTO E00 zO"O WD T | I I | dq I ! i] 1 i qT I T . Lo So 1 42 4 o A yi, Yo aft “ — G ee or a a 79 Y Pa 42 a / 48 / “ e ‘ ° = 0 Lo 1- UTW-SeTOU OT xX GO’ = A 46 . fe) , reTou ¢_OT ¥ zg €= $ Tal apt zeupAYyUTso1s4-q- TAZ a0 BOLO TYOTG-N ot f / | (UT) 4 / Jo sftsftoupséH 9u3 JO 40Td depo 3sdtd / | | L ama3tg sy Firure © Plot According to Integrated Slope Hi Equation - 2 tmmoLesemin LO.1 10,2 10.8 10.4 10.5 (sazou)°s c-OT¥9'E 4'E cE O'E g< ge hz 272 O's BT Qt tT e°t oO't af ‘© 4+ to +O | Oo se) -210- rs — 09 an e 0g | 00 apt zeipAYyU FsouhL-T- TAZ a0 e0 FO TYOTC-N ° jo sTsfktoupsy ey} 40g ~§ °SA °V/°s JO 401d G& eInZsty i” L Table 8 The & Chymotryvsin Catalyzed Hydrolysis Of The Ammonium Salt Of N-Acetyl-L-cysteic Acid Carboxylamide 1 Substrate Concentration = 200 u moles-:milliliter * Normality of Base used in titration = 0.10 N Milliliters of Base equivalent to total substrate in titration = 2,0 Initial ph = 6.20 Final pH = 6,18 T=0 T=24nr, T=84hr. T=22hr. T=274hr. End Point(ml.) 0.339 0.340 0.350 0.350 0.350 Initial pH = 6.85 Final pH = 6.79 T=0 T=24hr, T=8thr, T=22hr. Tse7shr. End Point(ml.) 0.398 O.414 0.420 0.425 0.420 Initial pH = 7.25 Final pH = 7.20 fl 32 1 T=0 T=23hr, T=83hr, ----- T=27shr. End Point(ml.) 0.255 0.200 0.385 0.350 Initial pH = 7.32 Final pH = 7,30 T=0 T=24nr, T=8shr. T=22nr. T=274hr. Ena Point(ml.) 0.390 0.400 0.400 0.420 0.420 Initial pH = 7.95 Final pH = 7.94 T=O T=24nr. T=84hr. T=22hr. T=27shr. End Point(ml.) 0.320 0.335 0.345 0.370 0.380 Initial pH = 8.30 Final pH = 8.25 T=0 T=2shr, T=8shr. T=22hr, T=27shr. End Point(ml.) 0.250 0.320 0. 324 0.350 0.355 H. T. Huang and Carl Niemann - J.A.C.S. 73 1541 (1951) The Kinetics of & Chymotrypsin Catalyzed Hydrolysis Of N-Acetyl-L-tyrosinhydrazide In The Presence of N-Acetyl- l-cysteic Acid-carboxylamide As An Inhibitor this (Tham Concentration = 0.5F for et of runs) tA initial Substrate Concentration = 6.67 x 1973 Molar Blank = 0.100 Extent of Hydrolysis = 6,234 Concentration of Inhibitor = 0.000 Molar & Dy (corr) — In(Dea -Dz) (Deo = Dy. 2 0,212 25.162 . 0.01247 4 0.380 24.994 0.01916 6 0.568 24. 806 0.02672 8 0.728 24 646 0, 03317 10 0.899 OA UT 0.04015 e 1,005 24 279 0.04819 14 1.258 24.116 0.05493 1 1.428 23.96 0.06392 18 1.582 23.784 O,07232 Vo = 2.41 x 10°? moles+min.” Initial Substrate Concentration = 6.57 x 1073 Molar Blank = 0.100 Extent of Hydrolysis = 7.04% 5 Concentration of Inhibitor = 10.26 x 1079 Molar % in D _ 7 D, (corr) In(D =D.) (D =D.) 2 0.209 3.22515 O,001ee2 4 0.390 3.21844 0.01941 6 0.618 3.20426 0.02859 8 0.809 3.20165 0.03820 LO 0.906 3.19405 0, 04350 Le Live 3.17835 0.05951 May 1.382 3.17755 0.05993 16 1,560 3.1700 0.06736 18 1.750 3.16248 0.07535 Vo = 2.63 x 1072 moles-min.71 4 * a 4 + Lo 1 7 a This column obtained by subtraction of the blank value fron the original data Tnitial Substrate Concentration = 4.23 pet We i Table 9° Continued Molar Blank = 0.0S0 Extent of Hydrolysis Concentration of Inhibitor 0.000 Molar Deo ’ D,.(corr) In(Dee -D, (Deo -D,) 2 0.153 2.76642 0.01605 A 0.280 2.75931 0.02316 6% 0.440 2.74912 0.033135 8 0.550 2.74206 0. 04041 10 0.669 2.73437 0.04810 12 0.810 2.72516 0.05731 14 0.916 2.71819 0.06428 16 1.086 2.70689 0.07558 18 1.1°7 2.69948 0.08299 1 1.79 x 197? moles.min. <j oO I Initial Substrate Concentration = 4.23 x 107° Molar Blank = 0.090 Extent of Hydrolysis = 7.50% Concentration of Inhibitor = 10.26 x 19073 Molar / % in Doe 1 D, (corr ) 1ln(Dee -D) | Deo -Dy 2 O.148 2.76764 0.01553 4 0.395 2.75200 ), 03047 6 O.415 2.75072 0.03175 8 0.535 2.74337 0.03910 12 0.830 2.72485 0.05762 14 0.930 2.71727 0.06520 16 1.064 2.70838 0.07409 18 1.220 2.69793 0, 08454 V., = 1.76 x 107> moles-min, 72 o x s 4 2 4. 2 rad - " Q This column obtained by subtraction of the blank value from che original data Table 9 Continued Tnitial Substrate Concentration = 2.53 x 1073 Molar Blank = 0.105 Extent of Hydrolysis = 6.75% Concentration of Inhibitor = 0.000 Molar . 1% in Deo t D, (corr) In(Dee -D,) (Dee -D,.) 2 0.075 2.24918 0.01931 dh 0.128 2.24411 0. 02428 6 0.237 2.23248 0.03601 8 0.396 2.22613 0, 04236 | 10 0.375 2.21758 0.05091 12 0.468 2.20629 0.06220 14 0.530 2.20055 0.06794 16 0.582 2.19477 0.07372 18 0.650 2.18718 0.08131 V_ = 1.05 x 107° moles-min.-1 Oo Initial Substrate Concentration = 2.53 x 1073 Molar. Blank = 0.075 Extent of Hydrolysis = 8.94% 3 Concentration of Inhibitor = 10.26 x 10 Molar % in Deo ‘ D, (corr) 1in(Deo -D,.) (Deo -Di) 2 0.119 2, 24887 0.01962 hy 0.161 2.243765 0.02470 6 0,280 2.23109 0.03740 8 0.363 2.22215 0.04635 10 0.416 2.21638 0.05211 12 0.519 2.20508 0.06341 4 0.601 2.19600 0.07249 16 0.675 2.18774 0.08075 184 0.777 2.17623 0.09226 V. = 1.16 x 1075 moles-min,71 Oo % This column obtained by subtraction of the blank value from the original data hae’ eg Lhe Table S Continued > * 4 4 4 2 s : _- 2 Tnitial Substrate Concentration = 1.69 x 10°" Molar Blank = 0,105 Extent of Hydrolysis = 7.13% Concentration of Inhibitor = 0.000 Molar % in Do ‘ Dy (corr): in(De -D,) (Deo =D) 2 0,048 1.84130 0.02429 dy 0.097 1.83322 0.03210 6 0.155 1.8239 0.04140 8 0.204 1.81506 0, 04936 10 0.245 1.81091 0.05441 12 0.304 1.79956 0.06576 1A 0.317 L.79741 0.06791 16 0.376 1.78759 0.06873 184 O.461 1.77326 0.08306 Vo = 6.85 x 10°° moles-min.” fH! Initial Substrate Concentration = 1.69 x 1073 Molar Blank = 0,100 Extent of Hydrolysis = 6.4% 5 Concentration of Inhibitor = 10.26 x 10° Molar . % in D 20 . D. (corr) 1n(Deo -D,) (Deo =D.) e 0.044 1.84246 0.02286 Mh 0.094 1.83450 0.03082 6 0.138 1.82747 0.03785 8 O.175 1.82148 0, 04384 nO) 0.243 1.81042 0.05490 Le 0.275 1.80516 0.06016 14 0.313 1.79888 0, 06644 16 0.386 1.78675 0.07857 18 O.412 1.78238 0.08294 6 4 V. = 6.08 x 107° moles-min.7+ * This column obtained by subtraction o the original data f the blank value Prom . ned . ae) initial Substrate Concentration = 1.27 x 10 ~ Molar Blank = 0.080 Extent of Hydrolysis = 7.41% Concentration of Inhibitor = O.000 la Doo t D.. (corr) In( Doo -D,) (Deo =D, 2 0.058 1.55033 0.02886 dL 0.075 1.5460 0.03312 6 0.120 1.53708 0.04211 8 0.173 1.52562 0.05357 - 10 0.193 1.52176 0.05753 12 0.258 1.50696 0.07223 14, 0.298 1.49806 0.08113 16 0.4305 1.40649 0.08270 184 0.358 1.48455 0.09464 Vo = 5.25 X 1078 moles+min, 71 Initial Substrate Concentration = 1.27 x 1073 Molar Blank = 0.110 | Extent of Hydrolysis = 7.58% Concentration of Inhibitor = 10.26 x 19073 Molar . % in Deo ‘ D, (corr) in(Deo -Dy) (Deo =D) 2 0.033 1.54773 O..03154 4 0.083 1.53704 0.04223 6 0.145 1.52401 — 0.05526 8 0.178 1.51798 0.06129 1c 0,212 1.51050 0.06877 12 0.253 1.50170 O.7757 14h 0.272 1.49717 0.08210 16 0.325 1.48523 0.09404 18 0.368 1.47545 0.10382 Vo 61 x 10 6 moles-min.~+ * a) Se ai » sy a This column opt the original dat -217- Table 10 The Kinetics Of Chymotrypsin Catalyzed Hydrolysis Of N-Acetyl-L-tyrosinhydrazide In The Presence Of Added Sodium Sulfate (Tham Concentration = 0.82F for this set of runs) Initial Substrate Concentration = 1.82 x 107 Molar Blank = 0.250 Concentration of Sodium Sulfate = 0,000 In D t. D,(corr)* in{D _-Dx) D -D,) 21/15 0.165 1.92161 0.01627 4 0.325 1.86295 . 0.04403 6 0.508 1.86191 0.07597 8. O.711 1.82986 0.10802 10 0.868 1.79940 0.13848 te 1.082 1.76849 0.16936 14 1.227 1.74344 O.19449 16 1.420 1.70729 0, 22336 18 1.600 1.69457 0.23069 Vo = 2.28 x 107° moles-min,~2 Initial Substrate Concentration = 1.82 x 1073 Molar Blank = 6.190 Concentration of Sodium Sulfate = 0.000 * in D t D, (corr) in(D -D,) (D -D,) 2 O.170 1.91513 0,02275 4 0,383 1.88114 0.05674 6 0.555 1.85395 0, 08393 8 0.774 1.81970 0.11918 10 0.955 1.78983 0.14805 12 1.130 1.76027 O.17761 14¢ 1.324 1.72633 0.21155 16 1.463 1.70129 0.23659 18 1.632 1.669097 0,26791 5 s, -L V, = 2.23 x 10 moles-:min. ie} * This column obtained by subtraction of the blank value from the original data bed on Table 10 Continued Tnitial Substrate Concentration = 1.52 x 19073 Molar Blank = 171 3 Sonnentvatlon of Sodium Sulfate = 1 x 10 ~ Molar 1% in Deo . ‘ D, (corr) In(Dee -D,) (Deo - ~D, } 2 O.1LTS 1.83780 0.10210 A 0.353 1. 80105 0.13683 6. 0.594. 1.76730 5 17058 8 0.776 1.73572 0: 20216 LO 1,012 1.69323 0, 24465 5 12 1.168 1.66412 0.27376 14 1.217 1.65479 0.28309 16 1.482 1.60282 0.33506 18 1.565 1.56716 O. 37072 V. = 2.52 x 1072 moles-min.74 io) ~ ee -2 ; Initial Substrate Concentration 1.82 x 1072 Molar Blank = 0.190 Concentration of Sodium Sulfate 5 x 1073 Molar iH} k D, (corr) In(Dee -D.) (We -D.) 2 0.169 1.91324 0.02464 A 0.371 1.88297 0.05491 6 0.582 1.85034 0.08754 8 0.793 1.81661 0.12127 10 0.971 1.78725 0.15063 12¢ 1.169 1.75354 0.18434 14. 1.311 1.72864 O,20g24 16 1.421 1.70802 0.22896 18 1.618 1.67162 0.26626 Vo = 2.56 x 107? moles-min.~+ x This column optained by subtraction of the biank value from the original data o I 2 2 2 Initial Substrate Concentration = 1.82 x 106 Blank = 0,200 5 Concentration of Sodium Sulfate = 10 x 107 Molar % in Deo ‘ D, (corr) in(Deoo -D,.) (D co De) 2 0,182 1.01132 0.02656 0.374 1.88251 0.05537 0.582 1.85034 0.08754 . 0.778 1.80270 0.13518 10 0.943 1.79193 0.14595 12 1.112 1.76336 0.17452 14 1.312 1.72846 0.20942 16 1.404, 1.69562 0, 24226 18 1.621 1.67203 0.26585 V, = 2.43 x 1079 moles-min. 71 4.2 , + 2 Initial Substrate Concentration = 1.82 x 107° Molar Blank = 0.170 Concentration of Sodium Sulfate = 40 x 1073 Molar x In. Deo t D, (corr) in(Dee -D,) Deo -D.) 2 -6.188 1.91043 0.02831 Hy 0.373 1.88266 0.05608 6 0.586 1.84971 0.08853 8 0.750 1.80730 0.12004 10 0.952 1.79076 O.147¢8 12 1.118 1.76140 0.17784 14 1.287 1.73289 (0.20535 16 1.448 1.70402 0.23422 18 1.598 1.67635 0.2618¢ Vi. = 2.51 x 1072 moles-min.71 x ; This column obtained by subtraction of the blank value from the original cata Vabple 10 Continued ~2 Tnitial Substrate Concentration = 1.82 x 10 lar Blank = 0,200 - Concentration of Sodium Sulfate = 50 x 107" Molar in Doo ‘ D, (corr) In(Deo -D.) (Deo -D,) 2 0.209 1.90733 0.03055 oT O, 436 1.87272 0.06516 6 0,623 1, 64288 0, 0ch00 8 0. B46 1,80796 0.12992 10 1.072 1.74432 0.19356 12 1,247 1.73904 O,1Le7o4 14 LALO 1.71091 0, 22703 16% 1.009 1.67429 0.26365 18 1.769 1.64384 0.29410 UT —_ O gL ane n * -1 o = 2.00 x 1O’- moles.min. Initial 82 x 1072 Molar Blank = 0.185 Concentratio on of Sodium Substrate Concentration = 1, Sulfate = 10 0 x 1073 Molar . D, (corr) in(Dee -D,) (eo Dy) at 0.195 1.90939 0. 02846 M. 0.353 1.88570 0.05215 6 0.515 1.86081 0.07707 8 0.688 1.83354 0, 10434, 10 0.866 1.80467 0.13321 12 1.046 L.7 7461 0.16327 14. 1.22 1.74397 0.19391 16 1.356 1.72060 0.21728 18 1.468 1.70220 22568 Vo = 2.25 x 1077 es emin, 7+ This column obtained by Subtraction of the blank value from the original data Sart ~22]- APPENDIX THE SYNTHESIS OF N-TRICHLOROACETYL-L-TYROSINAMIDE This compound is a desirable substrate to study because of its usefulness in providing evidence pertaining to the theory which relates the magnitude of ke and the molar volume of the N-acyl function. It is predicted that the compound will have the largest k. of any amide yet studied in this laboratory. ° At first it was thought that this compound could be formed by the acylation of tyrosinamide with trichloro- acetyl chloride. The reaction was attempted under the same conditions that tyrosine ethyl ester was acylated | (p.166). However the products which were obtained by this procedure had a low chlorine content. This was pre- sumably due to the fact that water very rapidly attacked trichloroacetyl chloride and prevented it from reacting with tyrosinamide. Under these conditions the trichloro- acetyl amide bond was also probably hydrolyzed. Next a nonaqueous acylation was tried. The conditions were Similar to those employed for the acetylation of cystine dimethyl ester dihydrochloride (p.171). The product from the reaction of one mole of acid chloride and one mole of amino acid derivative was analyzed and found to have a high chlorine content. This indicates that some O-N diacylation had taken place. Attempts to selectively hydrolyze the phenyl ester without cleavage of the trichloroacetyl amide linkage resulted in failure. | It was next proposed to protect the phenyl hydroxyl group by forming the carbobenzoxyl ester and then to acetylate under anhydrous conditions. The free phenyl hydroxyl group could then be regenerated by catalytic nydrogenolysis of the carbobenzoxy function. This idea was abandoned when it was found that under the conditions of the hydrogenolysis the chlorine atoms were cleaved from the trichloroacetyl rest. Thus when trichloroacetyl anilide fy was treated with hydrogen in the presence of a catalysis at atmospheric pressure, mono and dichloroacetyl anilide were formed. Finally it was determined to attemot the synthesis by the use of an amide-ester interchange. To this end ethylthio trichloroacetate was synthesized. The reaction was carried out in a Soxhlet Extractor. L-Tyrosinamide was placed in an extraction thimble, which was then placed in the upper portion of the extractor. A 20% solution containing 1.2 equivalents of ethylthio trichloroacetate and acetone was placed in the boiler. After one hour of operation all of the tyrosinamide was transferred to the boiler. The solution was avery dark brown color. Upon cooling, a crystalline mass settled out. This material was identified as L-tyrosinamide hydrochloride. No other material could be identified from ~223- reaction mixture. The experiment was then repeated using ethyl tri- chloroacetate in place of the thicester. Midway through the extraction a crystalline solid began separating out. At the conclusion cf the run, the solution above the crystalline solid was clear with no evidence of decomposi- tion. The crystalline material was recrystallized to a melting point of 184-185°C. It contained no chlorine. On the basis of the elementary composition it is suggested that this material is 2,2 dimethy1-5-(p-hydroxybenzy1)~ tetrahydroimidazole-4-one. This material presumably arises by a condensation of acetone with the tyrosinamide. Elementary composition Cale = 65.50 Calc 7.29 C H f Cale #2 12.72 N Found «- 12.27 (x]> = -18.2 (30% ethanol) The experiment was repeated a third time using an- hydrous ethyl acetate as the solvent. When the solvent was allowed to cool a new erystalline Substance separated out of a colorless solution. This material contained chlorine and proved to be the desired product. The yield of the crude material was 65% M.P. 158-160°C. The material was zed using chlorobenzene, methanol, water-methanol 3 D Q B me oe cr i) [4 Ie and anhy rdrous” ethanol-benzene as solvents, 9 O-C>~ch- “CHC -NH2, Ni ero M.P. = 160-161° c 2 . , [x 72° = +30.5° (30% ethanol) Elementary composition Cale = 41,89 Calc = 8.87 Cc N Found = 41.83 Found = 8.91 Cale = 3.38 Cale =33.79 H Cl Found = 3.3¢ Found =33.68 PROPOSITIONS Dialkylcarbodiimines have been shown to be active agents (1) in the synthesis of anhydrides. It is therefore pro- posed that they be used in the synthesis of tetra-alkyl pyrophosphites. \o) -OR he { 2 ROMP: -OH + RNGC=N-R ma RovP-O- Prog + RINHC NHR RO Tetra-alkyl pyrophosphites have been shown to be useful (2) but hereto- (3) as intermediates in peptide synthesis, fore have been prepared only in low yields. Thiol esters have been shown to possess a greater re- (4) on activity than the corresponding oxygen esters. particular their carbonyl group appears to be more sus~- ceptible to nucleophilic attack. It is therefore pro- posed that a synthetic route to alikyl acetylenic ketones lies in the reaction of thiol esters and sodium acetyl- (5) CH= CNa + C= O > | cH co? J—scyH= SCCR+ NaS Gis SCH SCLHs Dihaloisonitriles are compounds which can be con- sidered to be structurally analogous to phosgene. It is therefore proposed that these compounds offer a synthetic approach to dialkylcarbodiimines. PON=COL) HH,NR’ => R-N=CEN-R + 2H Ay wi e : ~226- Carbazole is an amine which displays acidic properties, The carbonyl group of N-acyl earbazoles(7) should then be subject to nucleophilic attack. It is therefore pro- posed that N-acyl carbazoles offer possibilities as mild, selective acylating agents. Heretofore the reaction of anhydrides with amines of alcohols has been followed by titrametric procedures , (8) It is desirable that a procedure be developed which is more convenient and of greater accuracy than the titra- metric procedure. It is proposed that hydroxylamine(?? offers the basis of such a colorimetric procedure. Con- centrated solutions of hydroxylamine may effectively quench the reaction and convert the anhydride to a hydroxamic acid. It has been shown( +0) that, under mild acidic condi-~ tions, the acyl group of N-acyl-&hydroxyl alkyl com- pounds easily migrates to the adjacent hydroxyl group. ( } ~C—=c- LH*] {oo T° qe Hom OH NuC-R Og-n Mia tt t is proposed that these "N to 0" shifts are important bd phenomena in the acid denaturation of proteins which contain serine or threonine. N-phthalamido-L~amino acid hydrazides could be of im- portant use in the field of amino acid chemistry. The reaction between the phthalamido group and hydrazine make these compounds difficult to prepare, (14) It has been indicatea!?? that by treating the phthalamido moiety with ammonia a reversible cleavage of the imide group > possible. a _ oe A. ah HR a me — 4 © It is © osed that the application of this reaction could greatly simplify the synthesis of N-phthalamido- L-amino acid hydrazides. It has been shown that chymotrypsin can cause the hydrolysis of f% keto acids. Cyclopentadiene reacts with aldehydes and ketones in the presence of base to form (13) highly colored fulvenes. It is therefore proposed that cyclopentadiene can form the basis of a colori- metric procedure which can be used to follow the chymotrypsin catalyzed hydrolysis of the compounds listed below. \l ul 9 9 Re Ce 2a . Nn a R- —E~CH2~ NO} "3 9. LO. ae DBR cate Acid chlorides are capable of interacting with other acids to form a mixture of acid chlorides and acias, 1"? It is therefore proposed that under conditions of high temperature and pressure a new industrial synthesis of acid chlorides could be developed using formyl chloride as an active intermediate, (19) HR+cz0 -= Hock Ron tHeD -—> R Bog + HCOoH It is proposed that the makers of "Sta Cold" are guilty of misleading advertising. = 229 — REFERENCES N. 8S. Corby, G. W. Kenner, and A. R. Todd - J. Chem. Soc. 1234 (1952) G. W. Anderson, J. Blodinger, A. D. Welcher - J.A.C.S. 74 5309 (1952) | A. EB. Arbusov and B. A. Arbusov - Ber. 65 195 (1932) a F, Lipmann - Amer. Scientist 43 37 (1955) J. A. Nievuland and R. R. 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