THE STABILITY OP VITAMIN A AND ITS ESTERS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University
By ALBERT JOSEPH PORLANO, B.Sc., M.Sc
The Ohio State University 1959
Approved by
^ Adviser Department of Pharmacy ACKNOWLEDGMENTS
I wish to express my thanks and appreciation to my adviser and friend, Dr. Loyd Harris, without whose assistance this study would not have been possible.
I would also like to thank the American Foundation for Pharmaceutical Education for their generous financial assistance in the form of a fellowship.
To my wife, Marie, who made my graduate studies have meaning by virtue of her kindness, cooperation and inspiration.
To Dr. L. M. Parks for his understanding guidance and council.
To all those who are too numerous to mention but are still near and dear to me, I give my heartiest thanks.
May, 1959 Albert ur. Forlano
ii TABLE OP CONTENTS
Page
Introduction ...... 1
Early History of Vitamin A ...... 2
Quantitative Determination of Vitamin A ...... 7
Introduction...... 7
Biological Method ...... 7
Spectrophotometrie Methods ...... 8
Chemical M e t h o d s ...... 13
Fluorescence M e t h o d s ...... lip
Vitamin A Alcohol ...... lip
Isolation and Chemical Characteristics...... lip
Ultra Violet Spectrum ...... 16
Infrared Spectrum ...... 17
Oxidation of Vitamin A ...... 17
Photochemical Oxidation ...... 17 Isolation and Characterization of Oxidation Products ...... 19
Effect of Solvents on Oxidation of Vitamin A . . . 22
Effect of Peroxides and Pro-oxidant Metals .... 2i|
Effect of Acidity and Moisture in Oils Upon Oxidation ...... 25>
Mechanism of Oxidation and Action of Antioxidants . 27
Spectroscopic Changes Occurring During Oxidation . 30
Miscellaneous Information Regarding Oxidation . . 30 Activity of Specific Antioxidants ...... 32
iii TABLE OP CONTENTS (Contd.)
Page
Compounds Related to Vitamin A ...... 32
Axeropthene ...... 32
Vitamin A A c i d ...... 33
Rehydrovitamin A ...... 33
Vitamin A Methyl E t h e r ...... 35
Retinene ...... 35
Vitamin A2 35 Anhydrovitamin A ...... 37
8, 9> Dehydrovit amin A ...... ij-3
Vitamin A E s t e r s ...... 1|1|.
Derivatives Other than Esters ...... 1|5
Isomerization in Vitamin A and R e t i n e n e s ...... Lf.6
Numbering Systems ...... i+6
Theories of Isomerization ...... Ip7
Neovitamin A ...... 5l
Chromatography ...... 53'
Biopotency of Isomers and Congeners ...... 58
Description and Purpose of Study ...... 59
Experimental Part ...... 61
Preliminary Studies ...... 61
Determination of the Stability of Vitamin A Palmitate in the Presence of an Homologous Series of Patty Acids ...... 67
Reagents 67
Decomposition Rates in "99%" Isopropyl Alcohol . 68
Preparation of Solutions ...... 69
iv TABLE OP CONTENTS (Contd.) Page
Rates of Degradation of the Vitamin A Ester . . . 70
Alumina Chromatography of these Samples ...... 73
Effects of Addition of Water to n99$" Isopropyl Alcohol Samples ...... 80
Rates of Degradation of the Vitamin A Ester .... 82
Alumina Chromatography of these Samples ...... 87
Paper Chromatography of these S a m p l e s ...... 90
Action of Isopropyl Alcohol, Water, Cyclohexane and Patty Acids in these Solvents on Anhydrovitamin A 98
Cyclohexane Systems ...... 98
"99$ " Isopropyl Alcohol Sy s t e m s ...... 106
"95$" Isopropyl Alcohol Systems ...... 108
1185$ 11 Isopropyl Alcohol Systems ...... 109 Paper Chromatography of the Isopropyl Alcohol Samples ...... 112
Preparation of Some New Esters of Vitamin A ...... 116 Introduction ...... 116
Synthesis ...... 120
Preliminary Data ...... 120
General Method of Preparation of the Esters . . . 122
Materials ...... 12I4.
Infrared Spectra ...... 127
Ultra Violet Spectra ...... 130
Determination of R^ Values ...... 132
Stability Testing of Esters ...... 136
Stability Against Base ...... 137
Saponification Equivalents of the Esters .... 138
v TABLE OP CONTENTS (Contd.)
Page
Stability of the Esters in 0.0113 N HCl - Anhydrous Ethanol ...... 139
Stability of the Esters in 0.0113 N HCl - Ethanol TJSP ...... lij.0
Stability of Esters in u99%" and "95%" Isopropyl Alcohol with and withoutPatty Acid ...... 1I4.I
Vitamin A A c r y l a t e ...... 1 )|?
Vitamin A Chloroacetate ...... II4J4.
Vitamin A Sorbate ...... 1I4.5
Vitamin A o( Chloropropionate...... 11^.7
Vitamin A Crotonate ...... 1)4.8
Paper Chromatography of the Reaction Mixtures . . II4.9
Discussion ...... 1J?2
Effect of Patty Acids ...... 152
Mechanism of Action of Patty Ac i d s ...... 153
Reaction Products ...... 155
Action of Water ...... 156
Mechanisms of Decomposition of Vitamin A Esters and Alcohol ...... 157
Summary and Conclusions ...... 162
Bibliography ...... 165 Autobiography ...... I7I4.
vi LIST OP TABLES
Table Page
1 . Conversion Factors for Vitamin A and Its Acetate • ...... 13
2. Extinction Values for Vitamin A Alcohol and Acetate ..... 16
3. Percent Vitamin A Destroyed by Aeration in One Hour ...... 22
’I4.. Stability of Vitamin A in Various Concentrations of Patty Acid . , ...... 25
5. Time Required for 20 Percent Loss of Vitamin A . 26
6. Extinction of Esters Before and After Saponification...... Ijl(.
7. Summary of Alumina Chromatography Systems . . . 56
8. Bioactivity of the Various Congeners ...... 56 9. Assay Values for Vitamin A in Various Ointment Bases ...... 63
10. Assay Values for Vitamin A in n-Hexane and "99%" Isopropyl A l c o h o l ...... 6l|
11. Rate Constants for Degradation of Vitamin A Palmitate in "99%" Isopropyl Alcohol Solutions 70
12. U.V. and I.R. Spectra of Chromatographed Fractions from "99%" Isopropyl Alcohol Systems 75
1 3 . Rate Constants for the Degradation of Vitamin A Acetate in "95%" Isopropyl Alcohol ...... 82
ll(.. Initial Rates of Formation of Anhydrovitamin A in Units of Vitamin A Lost in "95%" Isopropyl Alcohol ...... 83
15. Comparison of Total Vitamin A Loss to Conversion into Anhydrovitamin A in "95%" Isopropyl Alcohol ...... 81|
vii LIST OP TABLES (Contd.)
Table Page
16. IJ.V. and I.E. Spectra of "9$%" Isopropyl Alcohol-Vitamin A Acetate Control I4.5 0 . . . 88 N/30 Palmitic Acid h£>0 ...... 89 N/30 Caproic Acid 1|5° 89
17. R f Values of Chromatographed "95%" Isopropyl Alcohol Control Samples ...... 91
18. Rf Values of Chromatographed "95$" Isopropyl Alcohol, N/30 Caproic Acid I4.5 0 Samples . . . 92
19. Rf Values of Chromatographed "95$" Isopropyl Alcohol, N/3 0 Palmitic Acid i|5° Samples . . 93
20. Rates of Formation of Vitamin A Substance in Acetic and Caproic Acids with Anhydrovitamin A In Cyclohexane ...... 102
21. Rates of Transformation of Anhydrovitamin A into a Vitamin A Substance in "99%" Isopropyl Alcohol ...... 108
22. Rates of Transformation of Anhydrovitamin A into a Vitamin A Substance in "95%" Isopropyl Alcohol ...... Ill
23. Rates of Transformation of Anhydrovitamin A into a Vitamin A Substance in "85%" Isopropyl Alcohol ...... 112
21).. R f Values of Substances Pound in "99%" Isopropyl Alcohol Systems ...... 111).
25. Rf Values of Substances Pound in "85%" Isopropyl Alcohol Systems ...... 115
26. K„CL of Acids Related to this Study ...... 119 27. I.R. Carbonyl Peaks of New Esters (/i) .... 130
28. IJ.V. Extinction Coefficients of New Esters . . 131 29. Rf Values of Vitamin A Esters and Related Compounds ...... 136
30. Time Required to Consume 1 HI. 0.01N NaOH . . . 137
31. Saponification Equivalents of Esters ...... 138
viii LIST OP TABLES (Contd.)
Table Page
32. Rates of Elimination of Vitamin A Esters and Related Compounds in 0.113N HCl - Anhydrous Ethanol ...... IlpO
33* Rates of Elimination of Vitamin A Esters and Related Compounds in 0.0113N HCl-Ethanol TJSP . lljl
31}.. Rates of Decomposition of Vitamin A Acrylate in Isopropyl A l cohol...... ll+lj.
35. Rates of Decomposition of Vitamin A Chloro- acetate in Isopropyl A l cohol...... ll\$
3 6 . Rates of Decomposition of Vitamin A Sorbate in Isopropyl A l c o h o l ...... llj.7
37* Rates of Decomposition of Vitamin A OC Chloro- propionate in Isopropyl A l c o h o l ...... li|8
3 8. Rates of Decomposition of Vitamin A Crotonate in Isopropyl Alcohol ...... Ilp9
ix LIST OP FIGURES
Figure Page
1. Vitamin A Palmitate in "99%" Isopropyl Alcohol . . 71
2. Vitamin A Acetate in "95%" Isopropyl Alcohol . . . 71
3. Conversion of Vitamin A Acetate into Anhydro vitamin A in "95%" Isopropyl Alcohol ...... 85
ij.. Chromatography of N/30 Palmitic Acid-"95%" Isopropyl Alcohol b£>° Sample ...... 85
5. Chromatography of N/30 Caproic Acid-"95%" Isopropyl Alcohol l\S° Sample ...... 86
6 . Chromatography of "95%" Isopropyl Alcohol Sample . 86
7. Rates of Addition of Anhydrovitamin A to Caproic Acid in Cyclohexane at 50° ...... 103
8. Rates of Addition of Anhydrovitamin A to Acetic Acid in Cyclohexane at 50° ...... 103
9. Anhydrovitamin A in "99%" Isopropyl Alcohol . . . 110
10. Anhydrovitamin A in "95%" Isopropyl Alcohol . . . 110
11. Anhydrovitamin A in "85%" Isopropyl Alcohol . . . 113
12. I.R. Spectrographs of Chloroacetate Chloro- propionate and Acrylate Esters ...... 128
1 3 . I.R. Spectrographs of the Crotonate, Sorbate and Palmitate Esters ...... 129 llj.-l8,U.V. Spectrum of Vitamin A Chloroacetate, 133- o 19. Vitamin A Acrylate in "99%" Isopropyl Alcohol . . llj.3 20. Vitamin A Chloroacetate in "99%" Isopropyl Alcohol llj.3 21. Vitamin A Sorbate in "99%" Isopropyl Alcohol . . . llj.6 22. Vitamin A Chloropropionate in "99%" Isopropyl Alcohol ...... llj.6 23. Vitamin A Crotonate in "99%" Isopropyl Alcohol . . 150 x THE STABILITY" OP VITAMIN A AND ITS ESTERS INTRODUCTION During the half century that has elapsed since the recognition of accessory food factors, none has aroused more widespread interest than vitamin A As a result of the sustained effort of numerous workers in different laboratories, the main problems connected with isolation, structure and synthesis of vitamin A have been solved. It is, therefore, appropriate to review some of the principal achievements and problems that remain. A major problem is that of stability. Vitamin A was unstable in some preparations con taining vitamin A and fatty acids. The exact nature of this instability was undetermined (2) and it was believed that the fatty acid was responsible. This study was undertaken in an effort to determine whether the fatty acids caused this change and to learn the nature of their reactions. Only limited information on the action of fatty acids on vitamin A is found in the literature. 1 2 EARLY HISTORY The discovery of vitamin A was the outcome of fundamental studies on nutritional requirements. The existence of a factor capable of correcting an inability to see properly at night was recognized in early history. Aykroyd (l) mentioned that the Eber’s Papyrus recommended roast ox liver as a curative agent. Hippocrates also prescribed ox liver, and suggested it should be eaten raw after dipping it in honey. Modern studies have shown that livers of most animals are rich in vitamin A. The importance of a dietary factor in the pre vention of certain forms of night blindness, nyctalopia, has been learned through practical experience. Aykroyd (l) reported that Indians, in 1825 > realized that the affliction was caused by poor diet. The relation of vitamin A to dark adaptation was made by Frederica and Holm (3), who showed that "visual purple" could be formed only in the retinas. The investi gations of Wald (I4.) were mainly responsible for the recognition of "visual purple" as a protein complex of vitamin A aldehyde. Vitamin A was also connected with another ocular disturbance, which originated on the exterior surface of the eye, known as xeropthalmia. McCollum (5) cited the work of Magendie, who found that animals developed xeropthalmia when their diets were composed of materials deficient in vitamin A* 3 An early attempt to answer the question as to whether lipoids are essential for human nutrition was made by Stepp (6). A diet of untreated bread and milk was capable of supporting life in mice but if these foods were extracted with alcohol and ether before feeding, the animals died in a month. Death was prevented by restoring the fatty extract. Hopkins (7) mentioned finding "minimal qualitative factors" in whole milk which were necessary supplements to a diet of purified food stuffs if life and growth were to be maintained. Certain fats, such as butter and cod liver oil, had greater growth producing power than lard or almond oil. Egg yolk was found to resemble butter fat, but lard was ineffective (8). In 1915 McCollum and Davis (9) subdivided the vitamins into two groups: 1. Pat soluble A 2. Water soluble B The first chemical clue to the nature of vitamin A was found by the same workers (10) when they observed that vitamin A resisted the action of alkali and could be recovered in the unsaponifiable fraction after hydrolysis. In 1918 Steenbock et^ al^. (11) found that vitamin A was quite unstable when heated to 100° in the presence of air. It could be heated to 120° in the absence of air, for twelve hours without loss of potency (11). Zilva (12) found that oxidation by ozone caused a rapid loss of the vitamin. k The findings of McCollum et al. (llj.) and Osborne (1 5) that green vegetables such as cabbage, spinach, clover and grass all possessed vitamin A activity supported Steenbock's theory (1 3 ) that the bluish-green pigment chlorophyll concealed the presence of the yellow lipochrome pigments carotene and xanthophyll. Palmer and Eckles (16) proved that the pigments of animal fats were not produced in the animal but were absorbed from vegetable sources of pigments present in the diet. Vitamin A gave a bright purple color reaction with concentrated HgSOj^, which was a means of differentiating fish liver oils from vegetable oils. Drummond et al. (17» 1 8) correctly inferred that the purple color was an indication of the presence of vitamin A. The ability to form this color was lost when oils were heated in the presence of air. Since cod liver oil was not as intensely colored as butter it was believed that the vitamin A activity was not entirely due to carotenoids. This fact prompted two Japanese groups to attempt isolation of this unknown substance. In 1922 Takahashi (19) prepared concentrated forms of cod liver oil which he called "Biosterin" and claimed them to be pure vitamin A. He correctly concluded that the vitamin contained only the elements carbon, hydrogen and oxygen and that It was an unsaturated alcohol. In 1925 Drummond et al. (20) subjected the non- saponifiable portion of cod liver oil to vacuum 5 distillation and obtained pale yellow oils which were regarded as impure concentrates of vitamin A. The color reaction for vitamin A had the disadvantage that the color produced was very transient and difficult to reproduce. Rosenheim and Drummond (21) found that AsCl^ gave a color which faded too rapidly for matching. Carr and Price (22) reported that a saturated solution of SbCl^ in chloroform gave a reaction similar to AsCl^ in color and intensity but claimed better reproduce- ability. Duliere et al. (23) found that vitamin A and carotene could be differentiated spectrophotometrically or colorimetrically. The antimony trichloride reaction gave a blue maximum at 590mu with carotene and 608-612mu for vitamin A. In the U.V. region, vitamin A had a maximum at 3 2 0 -330mu which was not shown by carotene. Karrar (2lj.) established the structure of beta carotene by a study of the degree of unsaturation and oxidation products. Chemical methods similar to those used for carotene eventually led Karrar to the formula, ®20® 29^ * for vitamin A, which represents one-half the beta carotene molecule with a molecule of water added to form a terminal carbinol. The estimation of vitamin A potency was a difficult problem. Since the reliabilities of the SbCl^ reactions and the spectrophotometric methods were questionable, biological testing was the only suitable method of assay. 6 In 193I+. the Commission on Biological Standardization of the League of Nations (25) set 0.6ug of beta-carotene as equal to one unit of vitamin A activity and this is still the standard for provitamin bioactivity. Duping the past twenty years much work was done to determine a good vitamin A standard, which resulted in the adoption of 0.3l+l+ug of crystalline vitamin A acetate as equivalent to one •unit. Repeated attempts at crystallization of vitamin A. were unsuccessful until 1935 when Hamano (26) crystallized the beta-napthoate which proved to be biologically active. Two years later Holmes and Corbett (27) and Baxter and Robeson (28) crystallized vitamin A alcohol. The first claim of the synthesis of vitamin A was made in 1937 by Kuhn and Morris (29)• They obtained a few milligrams of bioactive material but their work was not reproducible. Ten years later Arens and Van Dorp (30), and Isler e_t al. (31) synthesized vitamin A. The conversion of beta-carotene into vitamin A in vitro was started in 191+5 hy Hunter and Williams (32) who found that H2O2 gave an 0.5 percent yield of retinene from beta-carotene. Meunier et al. (3 3 ) passed beta-carotene through a column of solid Mn02 and claimed 60-66 percent yield of vitamin A aldehyde which was reduced to the alcohol. In 1937 vitamin was found in fresh water fish by Lederer and Rosanova (31+) who recognized this substance because of its abnormal color reaction with SbCl^. 7 QUANTITATIVE DETERMINATION OP VITAMIN A The quantitative determination of vitamin A may be done by three principal methods: 1. Biological 2. Chemical 3. Physical Chemical and physical methods are complicated by the necessity of estimating two distinct groups of compounds, i.e., vitamin A and carotenoids which vary in chemical, physical and biological properties. Since there was no single method that could be relied upon to give the exact vitamin A potency, the analyst’s choice as to biological, spectrophotometric or chemical methods depended on the nature of the material. Spectrophotometric methods were the most accurate for estimating vitamin A in a rich source. When the vitamin A active substance was present in a weak source, biological methods were preferred and where the amount of active substance was intermediate, colorimetric methods were used. Biological Method The biological method for determining vitamin A or its provitamins depended on the measurement of the rate of growth in vitamin A deficient rats when they were given graded doses of the test material. The unit was fixed at 0.6ug of beta-carotene and 0.3ug of vitamin A alcohol. Most workers agreed that the two units were almost equal in bio logical testing (2 5). 8 Spectrophotometric Method Biological testa do not provide detailed information which is obtainable from physical methods. The nature of the substance causing the activity and the decomposition products could be determined, which was obviously a good starting point in attempts at stabilization. In 1931 Coward et al. (35) reported the determi nation of vitamin A by spectrophotometric and chemical methods on a series of eleven cod liver oils and later compared the results with biological tests. The spectro photometric results, based on the extinction at 328mu, were found to be more consistent with the biological results than were the chemical. It was expected that when suitable pre cautions were applied, the necessary conversion factor (C.P.) to relate extinction at 328mu with biological activity would be a constant. Consequently the extinction coefficient (E^ m ) ? was related to biological activity by a simple equation: En^ \ * C.P. = I.TJ./gm. lcm (328mu) Skilled workers could duplicate spectrophometric assay values with an accuracy of + £ percent, which was better than bio-assays. There was a question, however, as to how much of the 328mu absorption was due to vitamin A and how much due to the glycerides of the fat and to other substances contributing to the ‘'background” absorption. Removal of the glycerides by saponification helped remove some of the extraneous absorption. 9 Comparisons of the spectrophotometric assays were made with biological assays and a conversion factor (C.P.) of 1600 was adopted in 193^i- (2$), The standard for vitamin A alcohol and esters was changed to vitamin A acetate in 1914-8 (2 5). Chevalier and Chabre (36) concluded that a sharp maximum at 328mu was necessary for reliable spectrophoto metric assays, consequently research developed in two directions: (l) Attempts were made to determine a better C.P. for materials exhibiting a sharp maximum at 328mu. (2) Methods were sought for eliminating irrelevant absorption from concentrates before determination or by making appropriate deduction for their contribution to the absorption. Embree (37) recommended the use of amber glassware in the manipulation of these vitamins in order to minimize photodestruction manifested by a change in extinction. The absorption spectra of vitamin A was found to be considerably affected by the solvent used as a diluent (2 5). Smith et al. (3 8) and Gillam et al. (39) found the follow ing ratios of extinction for the same vitamin A sample in different solvents at 328mu; 100 percent in ethanol, 97.5 percent in cyclohexane, 97.8 percent in hexane, 107.5 percent in ether, and 89 percent in chloroform. Chromatographic separation was the first approach in the removal of undesirable constituents from the vitamin A samples (I4.O-I4.2 ). The adsorbent was chosen with extreme 10 care, since excessive activity caused the production of colored artifacts and loss of vitamin A activity. Glover et al. (ij.3 ) found that chromatography on bone ash allowed the removal of impurities without destruction of the vitamin. Morton and Stubbs (1)5) studied the absorption spectra of pure vitamin A acetate, using a Beckman spectro photometer. They found that the extinction at 313*&u was equal to the extinction at 3 3 8.3>mu and developed a correc tion factor to eliminate irrelevant absorption. In the U.S. Pharmacopeal Revision Committee recommended the use of vitamin A acetate as a standard because the cod liver oil standards were too unstable; a conversion factor of 189^ was accepted (I4.6 ). On the basis of the extinction of pure vitamin A, conversion factors were calculated by dividing the lcm into the total I.U./gm; therefore the C.P. for vitamin A: 3*300,000 I.U./gm. _ — *— 17!To'-/-£l— = 1900 in isopropyl alcohol The C.P. for vitamin A acetate: 2,900,000 I.U./gm. -,««« , , , — — 1—;■ .— — = 1900 in isopropyl alcohol 1525 The final concentration is calculated in this equation: I . U . Tnlrfo / \ n •*-. ™(corr.) x C.P. gm# o p ml • lcm These uncorrected C.P.s are only applicable to pure samples having a typical vitamin A absorption spectrum with a sharp maximum between 325 and 328mu. In order to allow for wider 11 use of this C.F., Morton and Stubbs (59) devised a method to correct irrelevant absorption based on the extinction at the maximum and at 313mu and 338.$ma. where pure vitamin A has 6/7 the absorption of the maximum. The irrelevant absorption can be calculated from the degree of flattening of the observed absorption curve as compared with the pure vitamin. This principle was based on an assumption that the substances causing the irrelevant absorption will form a straight line between 313 and 3 3 8.5mu, and that the line could have a horizontal, negative or positive slope. The negative slope was most prevalent, and will be considered first. A line was joined between 313 and 338.5mu where the extinctions should be equal and represent 6 /7 the extinction of the maximum. Any increase in absorption at the shorter wavelength over the longer wavelength will cause a negative slope. The correction was made in two steps: (l) A sub traction was made from the observed extinction at the maximum which was related to the slope of the irrelevant absorption line. (2) A further deduction was made to correct for the intensity of the irrelevant absorption at the lowest point of the slope. For example when cod liver oil was dissolved in cyclohexane, the three fixation points for this solvent were at 3 1 3 * 3 2 8, 3 3 8*5mia, and the extinctions at these wavelengths were 0.6ij.0, 0.712, 0.620, respectively. The total slope between 313 and 338.5mu was (E=0.6ij.0)-(E=0.620) = 12 E=0.020. The slope correction at 328mu was proportional to the distance of its position along the wave length axis. Therefore, (E=0.020) x (338.5 - 328) _ (E=0.020)x(l0.5) = 0.008 (338.£ - 313) 25.5 After this slope was deducted (correction step l), the extinction at 328mu became O.70I4.. The absorption at 328 and 3 3 8.5?mu now included the same flat irrelevant absorption called "x" and the absorption at 328mu was 7/^ that of 338,5mu after "x" had been deducted. Therefore, O.ffi - ?! = 7/6; x = 0.116 0.620 - x The correct E at 328 = (0 .7014- - 0.116) = 0.588. Elcm c0rrec^e^ multiplied by the C.P. will give I.U./gm; 0.588 x 1900 = 1120 I.U./gm. If this absorption had a positive slope the correction was calculated in the reverse direction and if the irrelevant absorption was horizontal, step 1 was omitted. This method has been integrated into a one step correction equation (ij.7) using isopropanol as the solvent (the fixation points are not identical with cyclo hexane ). E325 (corr.) = 7(E325 - E310) - I*.37$ (E310 - E33^) There are numerous references dealing with correction factors for free and esterified vitamin A in different solvents which are based on the principle described above. Boldingh et_ al. (lj.8) reported the following con version factors for different solvents. 13 TABLE 1 CONVERSION FACTORS FOR VITAMIN A AND ITS ACETATE . Vitamin A Vitamin A Solvent Alcohol Acetate Isopropanol 1825 1906 Ethanol 1825 1850 Cyclohexane 1906 1906 Light Petroleum (ij.0-60o ) 1825 1825 Chemical Methods These color reactions have been used qualitatively for the identification of vitamin A and its provitamin. The SbCl^ and similar types of reactions as the E^SOj^, Fuller’s Earth, AsCl^, guaiacol, phenol, HCIO^ and glycerol dichlorhydrin, were inaccurate because other congeners also produce the same colors (2 5). Some of the impurities exerted an inhibiting action upon the full development of color with vitamin A and SbCl^ and the color produced was transient. In concentrated samples the fading was neglected but with impure weak sources, the color faded within seconds. Even with these shortcomings, color reactions were suitable for weaker sources than could be used by spectrophotometry (25). Vitamin A forms a bright peacock blue color with SbCl^ having a maximum at 620mu and the oxidation products gave a red color with a maximum at 580mu. Heilbron, Gillam 1 1 1 . and Morton (ij.9) noticed that most oils produced maxima at 572rau and 606mu with SbCl^. More recently the maximum at 572mu was found to be due to oxidation products and the latter due to vitamin A. Fluorescence Peacock (50) made a detailed study of the yellow fluorescence shown by cod liver oils under U.V. irradiation and found that both yellow fluorescence and vitamin A activity were lost simultaneously when exposed to white light. The fluorescence, but not the bioactivity, was partially restored when oil which had been eagposed to light was kept in the dark for several months. Brocklesky and Rogers (51) suggested using fluorescence as a basis for quantitative estimations, by titrating the vitamin solution with maleic anhydride until the fluorescence was gone. Fluorescence had also been used for detecting the presence and location of vitamin A in chromatographic procedures. VITAMIN A ALCOHOL Isolation and Chemical Characteristics Vitamin A did not crystallize easily, even from almost pure concentrates. Consequently the physical characteristics such as M.P. and extinction at 328mu remained unknown even after Karrar et al. (52) obtained vitamin A in an almost pure form. Holmes and Corbett (27) prepared crystals of vitamin A alcohol by crystallization 15 from methanol in contact with dry ice for several days. The vitamin was obtained as pale yellow needles, M.P. 7 . 5 ° - 8 . 0 ° which were found to be a complex of vitamin A and methanol. Purification of vitamin A by fractional distillation under reduced pressure resulted in considerable loss of the vitamin (25)• With the use of molecular distillation the yield of vitamin A was considerably increased. Hickman (Sk-t 55) received patents for molecular distillation and for the "cyclic" still which is used today and eliminates many of the bad features of the older molecular still. Baxter and Robeson (28) isolated pure vitamin A from a 6£ percent concentrate which was crystallized from ethyl formate or propylene oxide at -35°. The resultant product was composed of pale yellow prisms having a M.P. of 6 2 ° - 6 3 ° and an extinction coefficient ( E ^ m ) of about 1 7 2 5 at 328mu. Vitamin A has the formula CgQH^OH. It is soluble in most organic solvents and fats, especially solvents containing hydroxy groups. It is insoluble in water, but can be dispersed by the use of the emulsifying agents as Tweens or Spans and by proteins. It was hypo- phasic when distributed between 83 percent ethanol and petroleum ether in the "phase test." The esters, on the other hand, have a tendency to be epiphasic as the length of the ester chain increases. The hypophasic nature of the vitamin A alcohol is due to its ability to hydrogen bond with the hydroxyl group of ethanol or water. 16 Since vitamin A contains no asymmetric carbon atom, it does not exhibit optical activity, however it does exhibit cis-trans isomerism due to the five conjugated double bonds. U,V. Spectrum Smith ei; al. (57) stated that a change in solvent will affect both the wavelength of maximum absorption and extinction coefficient in the U.V. region. Boldingh et al. (ij.8) reported the following values for vitamin A: TABLE 2 EXTINCTION VALUES FOR VITAMIN A ALCOHOL AND ACETATE Vitamin A alcohol Vitamin A acetate Solvent E E Isopropanol 325mu 52,300 325mu 5 0 , 2 8 0 Ethanol 32J?mu 52,14.80 325mu 5 1 , 1 8 0 Benzene 325mu 14-9,760 325m u 5 0 ,0 0 0 Light petroleum ether 325mu 5 2 , 0 0 0 325mu 5 2 , 0 0 0 Morgariedge (6 I4.) compared the spectra of vitamin A alcohol in polar and non polar solvents such as isopropanol and cyclohexane. He found the absorption maximum shifted from 325 to 328mu in the non polar solvents and there wa3 a corresponding decrease in extinction. This was due to the 17 Isopropanol association complex with vitamin A alcohol which enhanced light absorption. Extinction obtained in polar solvents should not be used interchangeably to correlate results between vitamin A alcohol and its esters. The esters, on the other hand, gave the same extinction in both types of solvents because there was very little complex formation. Infrared Spectra The infrared spectra of vitamin A and other related substances have been determined (60-63). Farrar (60) reported the infrared spectra of vitamin A, anhydrovitamin A and retinene. Vitamin A shows an assignment of unsaturation in the region of 900-1000 cm"-*-, Oroshnick (62) determined the infrared spectrum of vitamin A methyl ether. OXIDATION OP VITAMIN A Photochemical Oxidation Due to the large number of conjugated double bonds vitamin A was very sensitive to oxidation. Oxidation was first noticed in 192$, when Drummond ejb al. (6$) irradiated vitamin A with ultra violet light and claimed that the destruction was due to ozone formation. This conclusion appeared to be somewhat substantiated when Shrewsbury and Kraybill (66) exposed vitamin A to TJ.V. radiation in the presence of air. If the vitamin A was irradiated in an atmosphere of nitrogen, there was little destruction. 18 Meunler (67) treated an ethanolic vitamin A solution with U.V. light. He found that there was a decrease in extinction accompanied by precipitation. The same treat ment in hexane caused a shift in the absorption maximum but no precipitate and it was concluded that the oxidation products were insoluble in alcohol. This precipitated substance "A” had a maximum at 3l5mu and further irradiation of substance "A" caused the formation of a new compound with a maximum at 290mu. Chevalier and Dublotz (68) studied the photochemical destruction of vitamin A in slightly acidic alcohol when irradiated with U.V. light. Two new sub stances were produced; the substance "A" previously mentioned, and a substance ”B" which was described as a substance with an intense green fluorescence. In recent experiments Smith and Robinson (71) introduced new information which appeared to contradict the older literature. When vitamin A was irradiated for short periods in ethanol the absorption at 325mu initially decreased, but after a few hours of storage in the dark it was greater than before. Prolonged irradiation gave a smaller degree of recovery. Smith and Robinson believed that since vitamin A consisted of a mixture of geometrical isomers, the energy absorbed upon irradiation caused a change in the proportion of isomers. When the energy was removed, the equilibrium was slowly re-established. 19 Isolation and Characterization of Oxidation Products Investigators made little attempt to isolate specific oxidation products, consequently the exact nature of these compounds is still somewhat dubious. One of the first oxidation studies was performed by Euler et al. (£3) in 193I4.- They chromatographically isolated a substance on calcium hydroxide, having a Carr Price maximum at 580mu, which changed to 620mu on standing. Since it was believed to be a carotenoid, it was given the name "Hepaxanthin." Castle et, al. (10£) chromatographically isolated a fraction with an U.V. maximum near 270-280mu and a Carr Price color similar to that found by Euler. The oxidation product appeared as a constant component, consequently it was believed that vitamin A was composed of two isomers. They were0^,-vitamin A having a maximum at 270mu, and a beta-vitamin A, having a maximum at 328mu, the former having a SbCl^ maximum at 580mu and the latter at 622mu. The 0^. fraction was later identified by Karrar (106) as hepaxanthin. He synthesized hepaxanthin from vitamin A and thought the following formula to be possible. (a) It has a U.V. maximum at 275mu, with an Er-^x c x m = l$Q, with a SbCl^ it forms a maximum at 3>80mu. 20 The shift of the SbCl^ maxima from 580mu to 620mu was explained by the elimination of the epoxide and re formation of the original chromophoric system. Since the difference in absorption maxima between vitamin A and its epoxide was and the difference between carotene and its epoxide was 8mu, formula (b) was also considered possible, and he could not determine whether formula a or b was correct. Mallein and Javillier (108) prepared vitamin A epoxide by the oxidation of retinene. The furanoid form obtained by the action of HC1 on the oxidation product appeared to have the same spectra as the hepaxanthin de scribed by Karrar (106) with a U.V. maximum at 275?mu and a SbCl^ maximum at 570 shifting to 620mu. When the aldehyde group of 5»&-retinene epoxide was reduced, a new substance was formed with a maximum at 285mu in CHCl^. This indicated that the ©poxy group was on the cyclohexene ring rather than in the chain. Evidence was presented that a U.V. maximum in the region of 280-290mu was indicative of a chromophoric system with four double bonds in conjugation. Cormier (109) studied the action of zinc oxide on vitamin A epoxide by passing the latter through a column of activated zinc oxide. The zinc oxide removed the oxygen 21 from the ring to reform the vitamin A alcohol. The electrons, liberated through the removal of the oxygen, reformed the double bond. G-ormier (110) also found that hepaxanthin was formed when vitamin A was treated with MnOg. Embree and Shantz (ill) chromatographed a sample of fish liver oil and found a substance with a U.V. maximum at 290mu; with SbCl^ the maxima was at 617mu. They called this substance "Subvitamin A”. When it was treated with ethanolic HC1, a new substance was formed with maxima at 332, 3l(.8, and 367m.u (anhydrosubvitamin A). The same treatment for vitamin A produced maxima at 351> 371> 391mu. Anhydro- vitamin A was not adsorbed on alumina as strongly as anhydro subvitamin A. The latter apparently had a chromophoric group similar to anhydrovitamin A, but one less double bond in conjugation as indicated by the lower wavelengths of the maxima. It had an E ^ (290mu) = 150. lcm Subvitamin A was more strongly adsorbed on alumina than vitamin A. In distribution studies between petroleum ether and 83 percent ethanol, the ratios were as follows: subvitamin A 1I4.:8 6, vitamin A l|5:55» anhydrosuhvitamin A 50:50, and anhydrovitamin A II}.:8 6. Iso-anhydrovitamin A (l}.-ethoxy anhydrovitamin A) could easily be confused with anhydrosubvitamin A spectro scopically since both had maxima near 3 3 0 , 3 5 0, and 370mu. The greater polarity of the subvitamin A and anhydrosubvitamin A, demonstrated by the distribution coefficients, suggested that subvitamin A could be an 22 oxygenated derivative of vitamin A^ or vitamin A^. It has not yet been formed by oxidizing vitamin A in vitro. Effect of Solvents on Oxidation of Vitamin A The nature of the solvent had considerable effect on the type and degree of oxidation. Dann (69) studied the effects of different solvents on the oxidation of vitamin A, He concluded that many fats used as solvents were first oxidized and the peroxides that formed attacked vitamin A. The following table is a compilation of the results. TABLE 3 PERCENT OP VITAMIN A DESTROYED BY AERATION IN ONE HOUR Percent Percent Solvent oxidized Solvent oxidized ethanol 0 ethyl stearate 87 n-butanol 0 ethyl laurate 67 n-amyl alcohol 22 ethyl oleate 81 cetyl alcohol 0 triacetin 0 acetic acid 89 tributyrin 31 caproic acid Sk triolein 36 lauric acid 93 coconut oil 29 stearic acid 91+ peanut oil 60 oleic acid 92 20$ alcoholic KOH 0 ethyl acetate 0 23 The rate of oxidation of vitamin A was slow in alcoholic solvents, and it was concluded that alcohols exerted a stabilizing action against oxidation of vitamin A. Today these results might be questioned from the standpoint of the action of ethanol on vitamin A, since it is now known that ethanol reacts with vitamin A to form anhydrovitamin A. Although no oxidation had occurred, it seemed likely that some anhydrovitamin A may have formed. Vitamin A and anhydrovitamin A are indistinguishable by the Carr Price method which was used here. Holman (lOlj.) studied the co-oxidation of vitamin A and methyl linoleate by bubbling air through the system. Oxidation was accompanied by a decrease in extinction at 328mu, and an increase in the region of 235mu. When the log of the extinction at 328mu was plotted against oxygen uptake it followed a logarithmic function and all of the vitamin A was destroyed when 10 percent of the linoleate was oxidized. Battachyraya and Basu (99) studied the stability of vitamin A and its acetate in aqueous suspensions containing surfactants and concluded that it was less stable at a pH of 3 than at a pH of 6 . Hydroxy antioxidants were ineffective and the stability of the acetate was less than that of vitamin A. Therefore in aqueous solutions, vitamin A alcohol behaves differently than in oily solutions, where its stability is less than that of the ester. Coles et al. (7 2) found the same information and theorized that the surfactant molecules protected vitamin A from oxygen. 2k Battachyraya ejb al. (100) studied the stability of vitamin A alcohol, acetate and palmitate in vegetable oils, containing propyl gall&te and propyl gallate plus 0 .0 2 per cent citric acid. The controls without antioxidants were very unstable. The antioxidant and antioxldant-citric acid combinations reduced oxidation. Citric acid also acted as an antioxidant but in high concentrations it caused destruction of vitamin A. Effect of Peroxides and Pro-oxidant Metals In 1939 Smith (70) found that fatty acid peroxides caused destruction of vitamin A and that the peroxide value was an index of the stability of vitamin A in an oil. Vitamin A esters were considerably more stable against peroxide oxidation than vitamin A alcohol because it was believed that the hydroxyl group was necessary for oxidation. Roa (79) also found that there was a correlation between peroxide number and vitamin A destruction in an oil. The initial destruction was very slow at first but after the induction period was over vitamin A destruction proceeded rapidly. Roa (60) studied the catalytic action of metals on vitamin A and. found that their pro-oxidant activities decreased in this order: cobalt, manganese, copper, iron, cerium, magnesium, aluminum, zinc and lead; nickel and tin had little effect. The induction period in the presence of these metals was considerably reduced and peroxides did not accumulate. The reactions involved here were the 2 5 (l) formation of peroxides., and (2 ) decomposition of peroxides leading to vitamin A destruction. Peanut oil con tained 0 .009-0.012 ppm of copper and 0 .31-1 .1}. ppm of iron (8l). In these cases, citric acid acted as an antioxidant by chelating the trace metals. Addition of 2 ppm of iron or 0.2 ppm of copper irrespective of chemical form, destroyed vitamin A very rapidly (8l). Effects of Acidity and Moisture in Oils upon Oxidation Rhamnamurti et al. (82) studied the effects of acidity in shark liver oils exposed to air at 37°. The results are given in table I}.. TABLE 1}. STABILITY OP VITAMIN A IN THE PRESENCE OP VARIOUS CONCENTRATIONS OP PATTY ACID Percent Percent of vitamin A remaining in oleic acid 20 minutes 1±0 minutes 60 minutes 1 .1}. 85.7 71. k 50.0 3-k 71.1* 1*5*0 35.0 1*.2 57.1 35.7 25.7 5.3 35.0 ll*.3 5.0 10 .1}. nil nil nil The effect of ethyl gallate on preventing this re action was studied by determining the time needed for 20 percent loss. The results are given in Table 5* 26 TABLE 5 TIME REQUIRED FOR 20 PERCENT LOSS OF VITAMIN A Percent With oleic acid Control ethyl gallate 0.6 2 hours 5.75 hours 1.14- 1 hour 3 hours 2.3 I4.O minutes 1.3 hours 3 4 20 minutes 25 minutes U-.2 12 minutes 12 minutes 6.2 8 minutes 8 minutes The samples with high acidity showed the same rate of destruction in the control and in the sample with anti oxidant. This led to the conclusion that oxidation was not the only factor responsible for vitamin A loss when high concentrations of oleic acid were present. Bose et al. (8 3) studied the influence of acidity, moisture, and sunlight on antioxidant action in systems containing isobutyl gallate and citric acid. They found that free acidity and moisture in excess of 2 percent of each decreased the antioxidant action. In direct sunlight, the loss of vitamin A was great even in the presence of antioxidants. Fatty acids in fish liver oils increased the rate of oxidation when oxygen was passed through the system (9 3)• McGrillvary (91}-) found that the induction period was reduced to 1/160 of the original value when carotene was 27 aerated in liquid petrolatum or hydrogenated coconut oil in the presence of fatty acids. Mechanism of Oxidation and Action of Antioxidants Basu (8ij.) stated that unsaturated diluents for vitamin A increased vitamin A loss when the system was aerated. Antioxidants were effective in unsaturated solvents and in liquid petrolatum and their action was aug mented by glycerides. Since the stability of vitamin A in a natural glyceride system was the same as in the presence of an antioxidant, it was believed that the glyceride was needed for antioxidant activity. Basu (81}.) believed that the initial step of auto oxidation was due to the formation of a free radical by the loss of an ° < -methylene hydrogen ion and that the phenolic antioxidant prevented the formation of these free radicals and hydro-peroxides. Calkins (8£) showed that acid inhibitors of oxidation acted by donating protons, which regenerate the phenol from the phenoxide ion, thus insuring a constant supply of phenolic substance in the system. It was believed that a saturated diluent such as liquid petrolatum, would not enhance oxidation (81].). The effectiveness of antioxidants such as hydroquinone, phenols and phenolic esters were studied. Basu (81].) believed that the first step in the oxidation of vitamin A was due to the formation of a peroxide (III). 28 (I) HaOH (II) h*oh (III) The association of the antioxidant with vitamin'A caused a decrease in the concentration of antioxidant, thus the presence of a proton donating substance freed the anti oxidant from the complex and allowed it to continue to be effective. This was exemplified by the increased stability upon the addition of oleic acid to systems containing vitamin A in liquid petrolatum. When vitamin A was dis solved in a glyceride containing an antioxidant with no hydrogen donating substance present, the enhanced inhibition of oxidation was believed to be due to the release of fatty acids by hydrolysis of the ester. Basu (8i^) concluded that vitamin A alcohol might undergo prototopic change giving compound I which then joined with oxygen to form compound III. The presence of an antioxidant formed the complex (II), which regenerated vitamin A and the antioxidant in the presence of (H+ ). The replacement of the mobile hydrogen, by esterification, lowered prototopic change and the ability to take on an 29 electronic charge thus preventing the formation of type III peroxides. The antioxidant had no effect on peroxides that were already formed. Janecke (87) described the method of oxidation in the side chain MR” where conjugated double bonds are present: (1) R-CH = CH-CH2- => R-CH = CH-CH- (2) R-CH = CH-CH- + 0, =>• R-CH = CH-CH- U (1) R-CH = CH-CH- + R-CH = CH-CHg- — > R-CH = CH-CH- | - H +R-CH = CH-CH- ^ R CH = CH-CH- ^ I 9 The R-CH = CH - CH- can easily add oxygen and start a chain of autoxidation. Antioxidants may act by several mechanisms (87): 1. Agents that supply protons to neutralize the charge on the peroxide ion (R-OOH) which stops the chain reaction (acids). 2. Hydrogen donors which will tend to suppress protopic change and hydrogen capture (acids). 3. Agents which complex with pro-oxidant metals (chelating agents)• I}.. Agents which themselves are preferentially oxidized. 5. Substances that reduce the peroxide concentration (catalase). 30 Spectroscopic Changes Occurring During Oxidation Balomey (8 8) found that oxidation of vitamin A caused a decrease in absorption at 328mu and that the oxidation products had their maxima at shorter wavelengths. As the oxidation progressed the maxima shifted toward lower wavelengths in the following fashion: 328mu went to (312-310mu)-^ (29^-2 9 6)—^ ( 28I|.-286mu)— (27lj.-275mu ) and had maxima at 230mu and below 220mu. The extinction coefficient here was also constant at 290mu irrespective of the extent of oxidation. Halpern (89) found that during oxidation, maxima develop at 235mu and 275>-280mu and that the latter maximum was thought to be due to a new conjugated system with a smaller number of double bonds. Groot (91) commenced to work with some material, which he thought was pure vitamin A acetate, by dissolving it in various solvents. The solutions exhibited an absorp tion peak at 270mu which was not normal for vitamin A. Pure vitamin A solutions were aerated and subsequently developed peaks at 310mu, 275>mu, and below 220mu. Thus it was believed that the maximum at 270mu was due to oxidation products. Groot (92) also found that U.V. light caused maxima to be formed at 273> 280, and 310mu. The effect was shown to be oxidation because samples irradiated in the absence of oxygen did not show the characteristic peaks. Miscellaneous Information Regarding Oxidation Holmes et al. (8 6) developed a number called the ’’Antioxidant Index’’ which was determined as follows: 31 Antioxidant Index = Induction period of fat with antioxidant Induction period of fat without antioxidant It was theorized that the induction period was the time re quired for peroxides to form. Vitamin A and its oxidation products had the same absorption at 290mu called the "Isobestic Point” (77)* If it was desirable to know the original concentration of vitamin A in an oxidized sample it could be determined by the use of the following equation: El* (290mu) X 3000 = original vitamin A concentration. An lcm accelerated oxidation test involved treating the vitamin A with glass beads to increase the surface area exposed to air (73)• Adsorbents have also been used to increase surface area (7lj.)* A series of tests determined at Pfizer Laboratories from 1-18 days showed that gelatin coated vitamin A pellets, kept at elevated temperatures for 1200 hours, oxidized to the same extent as a sample kept at room temperature for 3 years under normal conditions (7 5) • Smith (76) placed vitamin A on filter paper in the presence of air and heat. The initial maximum was at 328mu, but after nine hours the maximum shifted to 310mu, and at 2ij. hours the maximum was located at 290mu. This treatment caused the following changes in vitamin A samples: 1. The bioactivity, at 328mu, Carr Price values, lcm and iodine values decreased. 2. Peroxide values decreased. 3. Concentration of aldehydes and ketones increased. I4.. Hydroxyl groups decreased. 32 Consequently Smith, thought that the double bonds of vitamin A are first oxidized to peroxide groups which then give aldehydes or ketones. Activity of Specific Antioxidants Kamath and Magar (93) rated the effectiveness of antioxidants in the following order: propyl gallate ^ NortLihydroguaretic acid di tertiary butyl p-cresols^ butyl p-hydroxyanisole. In the absence of oxygen these anti oxidants gave no protection against U.V. light, because U.V. light only caused cis-trans isomerization. N, n \ diphenyl p-phenylenediamine acted as a good antioxidant by chelation of pro-oxidant metals (101). Bose (7 8) studied acidic, phenolic and amine types of antioxidants and found that acidic antioxidants were not suitable because of the anhydro vitamin A formation. The phenolic antioxidants appeared to be best. Schlenk et al. (103) discussed the possibility of stabilizing vitamin A palmltate by making choleic acid complexes. COMPOUNDS RELATED TO VITAMIN A Axeropthene The hydrocarbon of vitamin A, called axeropthene or desoxyvitamin A, was produced by Karrar and Benz (112) and differs from vitamin A because hydrogen replaced the hydroxyl group. The spectrum in ethanol showed maxima at 331mu, 3l}.6mu, 36ijmu and the SbCl^ maximum was at 577m u . It had about 20 percent of the biopotency of vitamin A. 33 Vitamin A Acid Arens and Van Dorp (113) synthesized vitamin A acid which was not found naturally. It had biological activity without first being converted into vitamin A. The spectrum in ethanol showed a maximum at 3ij.3mu and it gave a red violet color with SbCl^ that was less intense than that produced by vitamin A (107). Vitamin A ethyl ester has been synthesized, and had a maximum at 350mu in ethanol. Rehydrovitamin A Shantz (lllj.) reported a substance called rehydro vitamin A, which was formed from anhydrovitamin A in vivo. The study of this conversion was prompted by the question as to. whether anhydrovitamin A had any bioactivity. Rehydro vitamin A, formed by hydration in livers of rats fed anhydrovitamin A, was reported to be 20 times as active as anhydrovitamin A. Chromatographic adsorption studies showed it was deposited in the liver in the esterified form. The spectrum in ethanol showed maxima at 35lmu and 369mu with an inflection at 330mu. This triple peak was similar to anhydrovitamin A, but the position of the maxima showed that one double bond was removed from conjugation. Since a double bond system in conjugation with a hydroxy group only had one maximum, the OH group of rehydrovitamin A was not in conjugation. Carbonium ion formation in the 3k presence of (H+ ). does not occur with rehydrovitamin A because the OH group is not activated by conjugation with an unsaturated system. Treatment with SbCl^, however, was strong enough to force the double bond system and OH groups into conjugation giving a blue color very similar to vitamin A and anhydrovitamin A. When Orosnick (116) attempted the synthesis of vitamin A methyl ether, only a small yield was obtainedJ however^ a product related to retrovitamin A (retrovitamin A methyl ether) was formed where the conjugated double bond system was pushed back one carbon atom all through the molecule. Compounds of this type are members of the "retro- ionylidene series" and have low biological activity. The U.V. spectrum of this compound had peaks at 3331 3i|8 and 367mu. Beutel £t al. (115) converted vitamin A and vitamin A acetate into retrovitamin A and its esters with aqueous HBr solution and formed maxima at 333> 3i+8, and 3&7niu. Under acidic conditions, vitamin A acetate was believed to be converted to the thermodynamically stable retrovitamin A through prototropic rearrangement by the conjugate acid, since polyenes were known to have proton accepting tenden cies . The release of strain in transition from regular to retro structure was shown by the shift in respective absorption maxima to longer wavelengths. 3 5 Vitamin A Methyl Ether Vitamin A methyl ether had a U.V. maximum at 326mu 1 a and a = 1660 in isopropyl alcohol. Vitamin A methyl ether gave a blue color with SbCl^, with a maxima at 620mu. The bioactivity is equal to 3 .0 X 10^ IU/gm, approximately 90 percent of vitamin A alcohol (116). The ether was more difficult to dehydrate with ethanolic HCl or toluene sulfonic acid than vitamin A alcohol. The stability against acid decomposition of the ether lies between the alcohol and acetate. Retinene Retinene has great importance in vision because it is necessary for the synthesis of rhodopsin by the retina. It is also important because it is theorized to be an inter mediary product in the formation of vitamin A from beta- carotene in animals. Ball (117) and Meunier (118) synthesized retinene by treating vitamin A alcohol with MnOg and KMnO^ respectively. Meunier (1 1 8) reported the physical constants for retinene: U.V. maxima; 3 8 5 ^ in chloroform, 386mu in cyclo- hexane, 36lmu in petroleum ether, and a Carr Price maximum at 66 ljmu. Cama (119) reported that retinenes 1. and 2 have sufficiently different chromatographic behavior to permit their separation on alumina columns. Vitamin A£ In 1931 Heilbron et al. (181) reported the discovery of vitamin A2 which gave a Carr Price maximum at 693mu and 36 U.V. maximum at 2\\.0-2>SOmx, The presence of the "693mu chromogen" in fish liver oils was reconfirmed by G-illam et al♦ (182). It was assumed that the compound contained 6 double bonds in conjugation. Farrar et al. (135* 183) confirmed the structure of vitamin Ag, by synthesis, as the dehydro derivative having an extra double bond in the beta-ionone ring and found that vitamin A2 was more susceptible to oxidation than vitamin A]_. Henbest et_ al. (121}., 132) found that anhydrovitamin Ag had a spectrum similar to anhydrovitamin A^ at 3 5 0, 3 6 8, and 390mu, whereas the SbCl^ maximum was at 693mu and that of anhydrovitamin A^ was at 620mu. It was more strongly adsorbed on alumina than anhydrovitamin A^. The infrared spectrum of anhydrovitamin A2 showed a band near llOO001"-1-, corresponding to an ether. The presence of an alkoxy group was demonstrated and the accepted formula on the basis of the evidence was 3-&lkoxy anhydrovitamin A-^. Vitamin Ag was easily dehydrated by treatment with N/3 0 HC1 in ethanol (1 2l|.). Methods of determining vitamin A^ and vitamin A^ in their mutual presence involved using the absorption differ ences produced by the Carr Price reagent; vitamin A had a maximum at 620 mu and the latter at 693mu (1 0 2 ). Balasundram et al. (9 8) fed rats anhydrovitamin Ag and the livers contained a substance with TJ.V. maxima at 33k> 3J+8, and 365mu corresponding to the spectrum of rehydro vitamin Ag. This situation was analogous to that when anhydrovitamin Ax was fed to rats (ill}.). 37 Anhydrovitamin A Edisbury et al. (137) made a blue complex of vitamin A with SbCl^ in CHCl^ which was easily destroyed by water. The U.V. spectrum of the new solution showed a decrease in the 328mu maximum and had other maxima at l4.25>mu, 399mu, 376mu, 357mu, 3l|.0mu, 32lpnu, 308mu, and 280mu. When vitamin A was treated with ethanolic HC1 there was also a decrease at 328mu with additional maxima at l).19mu, 392mu, 3&9mu, 3S>0mu, and 2>30mvL; the 3&9mu maximum was the most intense. The addition of base stopped the elimination reaction. With relatively dilute acids, 10“^- to 10“^ N the narrow bands at 392mu, 369mu, and 350rou appeared slower, approaching a maximum in 1 -2 days and disappearing there after. With higher concentrations of acid, N/5>0, the narrow bands were fully developed in 2>0 minutes but their rate of disappearance also increased because the substance re sponsible for these bands appeared to be very unstable. The critical factor appears to be the concentration of acid rather than the vitamin to acid ratio. The destruction of vitamin A could be prevented or stopped by the addition of sodium ethoxide to neutralize the HC1 , but this did not reverse the reaction. These workers had no concept of the formula possessing the triple maxima at 392, 3 6 9, and 350mu. Embree (138) isolated a substance from fish liver oils with triple maxima at 3i|8> 369, and 389mu. This sub stance with SbCl^ gave a blue color having a maximum at 62ljmu; vitamin A gives the same maximum when treated with 38 SbCl^. A similar material was made by treating vitamin A with N/3 0 HC1 in ethanol. It was believed that this substance was a "cyclized vitamin A." The author claimed it was a poor name because its structure had not yet been elucidated. He theorized that the anhydrovitamin A was formed from free fatty acids in the oil and attempted to prove this by treating the oil with 80 percent oleic acid at 200° for 3 minutes. The spectrum of the original oil changed completely, indicating a mixture of oxidation pro ducts and a small quantity of anhydrovitamin A. This appeared to be a poor experiment because the other constitu ents in the fish liver oil might have had some effect on the results. It also appeared that 80 percent oleic acid at 200° is an extremely rigorous treatment, and any conclusions would not be completely valid. When the same treatment was repeated with 10 percent oleic acid, very little "cyclized vitamin A" was formed. Meunier (139,li^0) believed the theoretical mechanism of the Carr Price reaction to be as follows: Shd3 OR BLUE COLOR 39 The addition of solvents containing negative ions destroyed the color by virtue of the loss of a proton at the I), position. Embree and Shantz (111), in 191+3» believed the reaction to be dehydration rather than cyclization. They prepared anhydrovitamin A by treating vitamin A alcohol with acid for 1 5 -2 0 minutes followed by chromatographic purifi cation. If the concentration of vitamin A was greater than 1 percent it favored the formation of orange-red polymeri zation compounds (li+l). The purified materials gave U.V. bands in ethanol at 35lmu, 371mu, and 392mu with = 2 ,5 0 0, 3 ,6 5 0, and 3 ,1 8 0 respectively and with SbCl^ it gave a color maxima at 620mu, E ^ ° = k.8 0 0. lcm If the anhydrovitamin A was allowed to remain in contact with HC1 in ethanol for longer than 12-26 hours isoanhydrovitamin A was formed. It differed from anhydro vitamin A by having absorption bands at 330, 350, and 370mu with the center band being the strongest and had values lcm of 1100, 1360 and 1100, respectively. By following the formation of isoanhydrovitamin A on a spectrograph, it was seen that the 392mu band disappeared as the 330mu band was obtained. The SbCl^ color reaction formed a maximum at 623mu. Isoanhydrovitamin A was more strongly adsorbed on alumina than anhydrovitamin A. The partition coefficient between petroleum ether and 83 percent methanol was the same as anhydrovitamin A, 97:3. Orosnick (ll+3) attempted to elucidate the structure of isoanhydrovitamin A. The absorption spectrum of this compound suggested that it was a lower vinylog of anhydro- vitamin A containing a retro chromophore structure with the alkoxy group. Chemical analysis showed that isoanhydrovitamin A was an ethyl ether, but was different from vitamin A ethyl ether which had a maximum at 325>mu. Anhydrovitamin A appeared to be stable in oil solutions but concentrates and pure preparations decomposed rapidly giving a rubbery polymerization product (lip). When vitamin A ester was refluxed with ethanol anhydrovitamin A, fatty acid and other products were formed. The anhydrovitamin A was separated from the other substances of the nonsaponifiable fraction by chromatography on alumina, calcium phosphate or magnesium oxide. The purified material did not give a positive Zerewitinoff reaction indicating the absence of hydroxyl groups (lJ+l). Since it reacted with maleic anhydride quickly, and did not have any cis peaks in the U.V. spectrum, anhydrovitamin A was assumed to be in the all trans form (llfif., ll}5 ). Shantz (lip.) stated that anhydrovitamin A was formed when vitamin A was treated with anhydrous alcohol. Meunier (ll|.0) described the theory of anhydrovitamin A formation, catalyzed by hydrogen ions: Anhydrovitamin A m The structure of anhydrovitamin A was finally elucidated (li+T) (II4-8) - Laughland and Phillips (ll|9) reported that vitamin A in contact with sodium bentonite produced anhydrovitamin A. Robeson and Baxter (130) reported that the extinction at 392mu would be a valid estimation of the anhydro vitamin A concentration using the following formula: Percent anhydrovitamin A = E ^ 392mu lcm______3180 An indication of the purity of anhydrovitamin A in ethanol can be obtained by measuring the ratios of these extinctions: E 351mu/371mu = 2500/3650 = 0 . 6 8 E 392mu/371mu = 3 1 8 0 /3 6 5 0 =0.87 Two papers regarding the preparation of vitamin A active substances from anhydrovitamin A have been reported. Embree and Shantz (150) treated anhydrovitamin A with acetic, propionic, butyric;, oleic, and benzoic acids in ethanol, methanol and non-alcoholic solvents. When alcohols were used as solvent, addition occurred forming a vitamin A ether with the alkoxy group being the same as the solvent. Where non-alcoholic solvents were used the vitamin A ester was formed corresponding to the carboxy acid used. Orosnlck (l5l) treated anhydrovitamin A with BF^ in methanol and obtained almost complete conversion to vitamin A methyl ether in 98 hours at 5°. When glacial acetic acid was used as the solvent, vitamin A acetate was formed. Petracek and Zechmeister (152) reported on the effects of treating anhydrovitamin A with BF^. They formed a complex which was easily hydrolyzed to yield an isomer of vitamin A, I4. hydroxy aexeropthene, where the OH group was on the cyclohexene ring. The U.V. spectrum was qualitatively that of vitamin A; however the infrared spectrum differed in the region of 9.75^ which was the region of -C-0- stretching because only the isomer had absorption at this wavelength. When partitioned between petroleum ether and 95 percent methanol the new polyene alcohol was less hypophasic than vitamin A. Since neovitamin A and vitamin A did not form anhydrovitamin A at the same rates with ethanolic HC1, Barholdt (153) studied the rates of dehydration of the various isomers with the idea of correlating the rates of dehydration with a particular stereochemical foim. Two features were considered: 1. The maximum value of ratio E 390/E0 maximum, (E° maximum = extinction at 326mu at time zero. E 390 = extinction at maximum for anhydrovitamin A at any time t). 2 . t maximum (time necessary to reach maximum ratio of E390/E0 maximum). As the concentration of HC1 was increased, the t maximum decreased. The maximum ratio for fi390/E° maxima, if all the vitamin A was converted into anhydrovitamin A, was calculated to be 1 .61 , from the following equation: X MW lcm390____ AA _ 3180 X 268 _ E390 E1 fo x m 1850 X 286 * E325 lcm325 vitamin A The catalytic rates of elimination of the acids used decreased in the following order: HC1, HBr, B^SO^, p-toluene sulfonic acid and acetic acid and the addition of a small amount of water decreased the rate. Budowski and Bondi (167) described a method of assay of vitamin A by conversion into anhydrovitamin A and measur ing the extinction at 391mu. 8, 9> Dehydrovitamin A Attenburrow et al. (15^4-) synthesized 8, 9 dehydro vitamin A with the following formula: This compound differs from vitamin A because it cannot easily be dehydrated by ethanolic HC1. The triple bond exerts a stabilizing effect which lends support to the idea that anhydrovitamin A formation involves a shift in double bond during dehydration. The triple bond renders Meunier's mechanism of the rearranged carbonium ion inapplicable in this system. Animal tests indicate a bio-activity equal to I4.O percent of vitamin A. It has U.V. maxima at 328 and 252mu. kk Vitamin A Esters Vitamin A esters demonstrated greater stability toward catalyzed dehydration and oxidation than vitamin A alcohol. Mead (156) prepared the stearate, diphenyl acetate, Ij. nitrobenzoate, acetate, anthraquinone-2 carboxylate and 2-napthoate. The highly purified esters appeared to deteriorate on standing even in the absence of oxygen, however the crystalline 2-napthoate and anthrazuinone-2 carboxylate appeared to be most stable* In 191J-2, Baxter et al. and Milas (155* 163) reported the synthesis of the acetate, beta napthoate, palmitate and succinate. The acetate showed the best resistance against oxidation. The following table shows the values before lcm and after saponification. TABLE 6 EXTINCTION BEFORE AND AFTER SAPONIFICATION OF ESTERS (E1^ lcm ) Ester Acetate Palmitate Succinate b-napthoate before lcm saponification 1510 9i|.0 12)4.0 1090 1710 1700 1700 1700 SbCl- E1^ 14.580 291^.0 ■i lcm 2535 These esters were synthesized by treating vitamin A alcohol with the acid chloride in a non reactive solvent. Pyridine was generally used as a proton scavenger to inhibit any anhydrovitamin A formation (155* 158). Isler et al. (158) studied the kinetic rates of hydrolysis of various esters in base. He found that vitamin A acetate was hydrolyzed most rapidly and the palmitate most slowly. Beutel (159) described methods of making vitamin A palmitate with palmityl chloride in pyridine; the impurities as palmitic acid and palmitic anhydride were removed by ion exchange columns• Garibaldi (160) reported the synthesis of the water soluble biologically active glycerophosphate ester .0 CHp-OH R—0-P<“ ----0-CH OH CH2-0H Derivatives other than Esters Bernhardt at al. (161) reported that biliruhln and biliverdin exerted a stabilizing action on vitamin A in vitro by forming complexes. These were tested in aqueous solutions at a pH 8.5 with air bubbled through. Klein (162) reported the synthesis of vitamin A amine and claimed a bioactivity of 50 percent that of vitamin A. It had a TJ.V. spectrum similar to vitamin A because of the same chromophore group. The infrared spectrum showed no -OH group but did show either a primary or secondary amine. kb Lunnel (l6ij.) reported the structure versus activity relationships of vitamin A type compounds synthesized up to this time. When phenyl groups replaced the beta ionone group, activity was lost; therefore the polyene side chain was not enough to confer vitamin A activity. Isomerization in Vitamin A and Retinenes Hubbard and Wald (121) showed that cis-trans isomerism was essential for the formation of rhodopsin. All trans retinene does not combine with opsin; however the 2,Ip di cis isomer was successfully converted to rhodopsin and was designated as neoretinene b. There has been considerable confusion about the numbering system for these isomeric aldehydes and alcohols* Wald (122) consolidated the information on the 3 numbering 3' Geneva System Present System CHO Hubbard and Wald System (122) k l The discussion of stereoisomerism is also applicable to vitamin A and its esters. Only four stereoisomers were originally expected: all trans, 3 cis, 3*5 di-cis, and 5 cis (H-W system), however Hubbard and Wald showed the presence of five stereoisomers (121). Trans retinene had a maximum at 383mu, the cis isomers had maxima in the region of 376- 377.5mu and the di cis isomers have maxima in the region near 369mu indicating the displacement in ethanol was about 5 .5-7mu per cis linkage. All five isomers gave the same Carr Price maximum at 666mu. Vitamin A was isomerized with heat (125). The equilibrium mixture invariably contained a 2:1 ratio of trans/cis isomers. Isomerization proceeded well at 100° and slower at lower temperatures. Iodine was found to be an effective catalyst for isomerization of retinene isomers and vitamin A alcohols (123). Melting of crystals also causes isomerization (126). Since all of the isomers produced the same substance with SbCl^* it supported the theory of Meunier and Vine (191^-7) who suggested that the blue color of vitamin A plus SbCl^ was due to a vitamin A cation and that the greatly increased resonance that accompanied carbonium ion formation was responsible for the blue color. The increased resonance forced the molecule into the all trans form which offered the least interference to resonance. It was originally believed that only the methylated carbons at 9 and 13 could assume the cis configuration because at the other double bond positions the cis configur ation would encounter serious steric hindrance causing a large departure from coplanarity. A large twist in the molecule would interfere with resonance, consequently making the hindered cis forms very unstable, thus only the four isomers were expected. It has been found that there was a fifth isomer: neo retinene b (121, 122). It was assumed that neo retinene b must have a hindered cis linkage since the infrared spectrum of this molecule shows such a linkage. This type of link can only occur at the 7 or 11 position. n Example 9,13 cis Example 11 cis Example 7 cis Fig. A Fig. B Fig. C Even unhindered cis linkages caused some overlapping of hydrogen atoms as shown in Figure A. The slight twist in the molecule caused a partial block in resonance with a slight fall in extinction and displacement of the U.V. maxima toward the shorter wavelengths as compared to an un hindered cis linkage. Links of the 11 cis type (Figure B) were more strongly hindered through overlapping of hydrogen and methyl group. The completely hindered cis linkage (Figure C) In which 3 methyl groups overlap involved a large degree of hindrance. ^9 Hindered neoretinene b had the lowest U.V. extinction. The cis isomers all showed three absorption bands. The major one was in the region of 380mu and 2 cis peaks in the region of 255mu and 260mu. The unhindered cis isomers followed the general rule of spectra; the trans showing the highest extinction followed by the 13 ds,9-13 di-cis, 11 cis, and 7 cis last. The corresponding stereoisomers of vitamin A dis played similar relationships except that neovitamin A (13 cis) had a maximum 3i*iu higher than the all trans alcohol, which appeared to violate the rules for stereoisomers. A comparison of the neovitamin A and transvitamin A formulas showed that the only difference was in the position of the H and the OH groups; therefore the length and shape of the molecules were not altered. Consequently, there was no reason why a terminal cis linkage should induce the effects of an internal cis linkage. The increase in wavelength of the maxima is an indication of a terminal cis linkage in vitamin A series. Neoretinene A, however, displays the ordinary characteristics of cis linkages. In this case the cis linkage bends the chain causing electronic vectors at right angles to the main axis of the conjugated system. Therefore, a perpendicular component was added along the axis to the main mode of oscillation, cuasing the formation of a cis peak at a shorter wavelength with smaller ex tinction than the main peak. The presence of the cis peak, therefore, was an indication of bending and its height was a 50 measure of the degree of bending. A single central cis peak link caused the greatest degree of bending and consequently the largest cis peak. Two cis links oppose each other by bending the molecule in opposite directions, thus having a tendency to straighten it out giving lower cis peaks. The absorption maxima at the shorter wavelength was called a subsidiary cis peak. With the hindered cis links, distortions were found in the absorption spectra. The main absorption band lost the fine structure of the all trans isomer and was displaced toward the shorter wavelength farther than the unhindered cis isomer. The additional peaks in the TJ.V. induced by the partial chromophores were induced by the break in coplan arity. Neoretinene b has a subsidiary peak higher than the main peak, the latter having the smallest wavelength maxima of all the isomers. The spectrum of trans vitamin A was degraded as compared with a straight chain conjugated pentaenes. Vitamin A has lost its fine structure and the maximum was displaced toward shorter wavelengths. It had a low extinction due to steric hindrance between the 1 methyl and the 8 hydrogen which forces the beta-ionone ring out of coplanarity and the double bond was only partly conjugated with the side chain which only makes a small contribution to the chromophore. Cis isomers represent higher energy states than the trans isomer; the hindered cis still greater. 5 1 Neovitamin. A Robeson and Baxter (12?) crystallized neovitamin A from the mother liquor of vitamin A alcohol. It had a M.P. of 59-60°, a maximum at 328mu, = 1675 &n vitamin A had a slower rate of oxidation and dehydration in ethanolic HC1 than vitamin A alcohol (127, 129). Since maleic anhydride reacted much faster with vitamin A than with neovitamin A, and since neither adduct reacted with SbCl^ to give a blue color, Robeson and Baxter (130) used the different rates as a means of distinction between the two forms of vitamin A. The different rates of addition and properties of these adducts enable the isomeric vitamin A's to be separated chromotographically (1 3 1 ). Meunier et al. (129) stated that vitamin A and neovitamin A differ from each other in the configuration of the double bond closest to the carbinol group. The infrared spectra of neo vitamin A and vitamin A were almost identical (130). Neovitamin A had higher absorption in the region of 200-280mu and 330-390mu and had a cis peak near 250mu. When neovitamin A was irradiated with U.V. light, there was an initial increase in absorption at 325mu probably due to the formation of the all trans vitamin A. The later effects were a decreased absorption at 325mu followed by a new peak at 275mu due to oxidation products (probably hepaxanthin) (130). 52 Neovitamin A esters are more difficult to saponify than vitamin A esters (130). Dehydration of neovitamin A in ethanolic HC1, formed a substance which was identical with anh.ydrovitaniin A. This showed that when anhydrovitamin A was formed it changed to the all trans form (130). Vitamin A could be separated from neovitamin A by the use of a sodium alumina silicate column since the latter is less strongly adsorbed (130). Harris, Ames, and Brinkman (133) determined that neovitamin A had approximately 8j? percent biopotency of vitamin A. Rats had the ability to convert either isomer into an equilibrium mixture of 88 percent trans and 12 per cent neo form. Robeson et al. (13I4.) (using the Geneva system of numbering) discussed the properties of the different isomers. Neovitamin A: M.P. 580 - 60° (maximum at 328mu) reacted slowly with maleic anhydride; trans: M.P. 62-61f°, maxilma at 325>mu, reacted faster with maleic anhydride; 2,6 di cis M.P. 58°-59°, maxima at 32i|mu, reacted slowly with, maleic anhydride; and 6 cis M.P. 81° - 82°, maxima at 323mu and reacted faster with maleic anhydride. The cis isomers all show a cis peak in the region of 250-260mu. Only the double bonds in the 2 and I4. positions reacted with maleic anhydride. After the adduct was formed, the typical vitamin A curve was destroyed and replaced by a curve with maxima at 233 and 265mu (I3i|.). 53 Using the "present" system of numbering, Plack (136) defined the various isomers of vitamin A and studied their reaction rates with maleic anhydride as a continuation of the Robeson and Baxter method (130) where a mixture of vitamin A and neovitamin A were determined, but Plack also studied other isomers. He determined the rates of reaction of the different isomers in an attempt to correlate rates with a specific isomer. CHROMATOGRAPHY The chromatography of vitamin A and its precursors was first reported by Palmer and Karrar (165, 1 6 6 ). It was found that the hydroxy carotenoids were more strongly ad sorbed on alumina and calcium carbonate than vitamin A. In 1938 a comprehensive chromatographic study was made by Castle et al. (105) on a purified halibut liver oil concentrate, using alumina and calcium carbonate columns. It separated into five main zones, listed below in descending order: 1. At the top there was a zone with strong maximum between 270-280mu, with SbCl^ the maximum changed to 580mu indicative of oxidized vitamin A. 2. A zone which was rich in vitamin A-,maximum at 328mu. 3. A deeply colored zone containing vitamin A. The spectrum demonstrated a mixture of free vitamin A and poly merized vitamin A. 51+ 1+. The next zone appeared to be a mixture of and beta vitamin A (C< vitamin A had a maximum at 280mu; beta vitamin A had a maximum at 328mu). 5. A yellow zone least strongly adsorbed, had maxima at 31+8, 369, 389mu and was believed to be anhydro vitamin A. Datta and Overell (168, 169) used alumina impregnated filter paper as a means of separating vitamin A, vitamin A esters, anhydrovitamin A, retinene, and other chromogens. Holmes (170) found that orange-red colors developed sometimes when vitamin A was chromatographed on an alumina that was too active. No attempt at identification of this colored substance was made, however the importance was that adsorbents that were too active could affect vitamin A. Alumina may be deactivated by the addition of water. Meunier and Vinet (ll+6, 171+) reported some of the characteristics of this new pigment formed. It had maxima at 330mu and 1+3Omu and a SbCl^ maximum at 589mu and was thought to be a di vitamin A ether formed by the extraction of a molecule of water from 2 molecules of vitamin A. Brown (171) used filter paper impregnated with a silicone and a 60 percent acetonitrite - 1+0 percent water eluant. The order of increasing R^ values were palmitate, acetate, alcohol, and oxidation products. Kaiser and Kagan (172) modified the paper chroma tography method by using untreated filter paper and 50 percent water isopropyl alcohol as an eluant. The values 5 5 were vitamin A palmitate, 0.00; acetate, about 0 and alcohol, about 0.95* The presence of a surfactant caused the palmitate to move as fast as vitamin A alcohol. Reverse phase partition paper chromatography was used in the separation of vitamin A substances (173)- The paper was impregnated with hydrophobic substances such as mineral oil, vaseline, olive oil or paraffin; polar solvents, such as ethanol, methanol, or aqueous mixtures of these alcohols were used as the eluants. The following R^, values were reported; alcohol 0.978, acetate 0.537> and palmitate O.O9I4.. It was observed that increasing the length the MR" groups of the ester decreased the R^ values and increasing the number of polar groups on the compound increased the Rf values. Eden (175) reviewed the column methods for separating free and esterified vitamin A and the results are presented in Table 7» 56 TABLE 7 SUMMARY OP ALUMINA CHROMATOGRAPHY SYSTEMS Solvents for Solvents for Adsorbents Vitamin A Ester Vitamin A Alcohol A12°3 Hexane Ethanol A1203 2$ acetone-hexane 8fo ethanol-hexane a i 2o3 i|$ acetone-light increasing concentration petroleum ether of acetone in light petroleum ether a i 2o3 light petroleum 20fo acetone-light ether petroleum ether Bone Meal 2.5$ CHC1 -light acetone petroleum ether Silcic Acid light petroleum chloroform ether MgO Celite light petroleum 5$ CHClo - light petroleum ether ether Vitamin A alcohol on alumina columns traveled with hydroxy carotenoids whereas esterified vitamin A was less strongly adsorbed and moved with the hydrocarbon carotenoids. It appeared that increasing numbers of OH groups in the molecule increased the strength of adsorption. Oxidation products were most strongly adsorbed. Hydroxylated vitamin A compounds were not removed by nonpolar solvents such as petroleum ether or cyclohexane. Swain (176) studied seventy-one materials in conjunction with their ability to adsorb vitamin A and found 57 that salts did not adsorb vitamin A, clays destroyed vitamin A, CaO and MgO caused some destruction and alumina, calcium hydroxide, calcium phosphate, silicic acid and sugar were rated as good adsorbents. Bro-Rassmussen and Hjarde (177) described a method of separation of vitamin A, neovitamin A, and vitamin Ag using a dicalcium phosphate column. The order of increasing strength of adsorption was neovitamin A, vitamin A, and vitamin Ag. Barnholdt and Hjarde (178) chromatographed a 2:1 mixture of vitamin A2 and neovitamin A^ using a dicalcium phosphate column. It appeared that the neo form was more easily eluted than trans form. Keuning et al. (179) devised a method for determin ing the activity of adsorbents by a "Shake Test," using a solution of the compound to be chromatographed. This Shake Test gave a good idea as to the suitability'of a combination of adsorbent and solvent. The best eluant can be determined by measuring the amount removed from a solution of that substance by the adsorbent. Gillam et al. (180) used reverse phase partition chromotography in the separation of vitamin A and decompo sition products. The column consisted of a non wetting Kieselguhr as a holder for n-hexane; 60 percent water methanol was used as the developer. BIOPOTENCIES OP ISOMERS AND CONGENERS Moore has compiled a summary of the bioactivities of various congeners which are listed In Table 8. TABLE 8 BIOACTIVITY OP THE VARIOUS CONGENERS (2^) Approximate Process Product Activity All Trans A = 100$ Esterification Natural or artificial 100$ esters Oxidation Aldehyde 100$ Cis Isomerism Cis Isomers 23 - 1$ % Ether formation Phenyl or methyl 10 - 100$ ethers Dehydrogenation Vitamin A2 30$ Loss of oxygen Axeropthene 10$ Ketone formation C2i Ketone 10$ Demethylation Norvitamin A 3$ Addition of CH2 Homovitamin A 1 .5$ Dehydration Anhydrovitamin A 0.ii$ Condensation Kitol 0$ Oxidation Epoxide 0$ Hydrogenation Dihydrovitamin A 0$ Oxidation Vitamin A acid 50$ 59 DESCRIPTION AMD PURPOSE OP THIS STUDY" At the time this study was initiated, there was considerable interest in a cosmetic preparation containing vitamins A, D, E and panthothenyl alcohol in a vanishing cream base. When this product was subjected to stability testing, it was found that much of the vitamin A potency deteriorated. The exact cause of this decomposition was not determined. Since it was known that vitamin A was very sensitive to strong acids, it was assumed that stearic acid was the causative agent. The rate of decomposition was not as fast as that produced by a strong acid, consequently it was believed that the rate of decomposition was proportional to the small (H+ ) produced by the stearic acid. No compre hensive study was conducted at that time to determine the exact nature of the vitamin A decomposition in this preparation. A search of the literature did not show any information concerning the action of an homologous series of fatty acids upon the stability of vitamin A so a study was undertaken. The rates of vitamin A decomposition in isopropyl alcohol with and without fatty acid were measured. The chromatographed reaction mixtures elucidated the presence of various decomposition pathways such as hydrolysis, oxidation, and elimination. Samples containing fatty acids appeared to maintain a higher concentration of vitamin A at all times, 60 however these samples contained less anhydrovitamin A. It was concluded that the fatty acid reacted with anhydro vitamin A to reform a vitamin A substance. This aspect was subsequently studied by measurement of the rates of vitamin A substance formation when anhydrovitamin A was treated with fatty acids solutions in Isopropyl alcohol and a control containing only solvent. It appeared that vitamin A was very sensitive to eliminative decomposition, consequently the author believed it would be desirable to synthesize esters which would theoretically be resistant to acid attack and eliminative decomposition. In these new esters the acid radical contained a chloro group in the 2 position or 2, ij. unsaturation. After these esters were synthesized their stability was determined in the presence of the following reagents: 1. Base-ethanol USP 2. HCl in anhydrous ethanol 3. HCl in ethanol USP 1+. "99%" isopropyl alcohol 5. "95%" Isopropyl alcohol 6. a fatty acid in "99%" ana "95%" isopropyl alcohol 61 EXPERIMENTAL PART Preliminary Studies A preliminary study was conducted in which, vitamin A was dissolved in a variety of ointment bases and liquid solvents such as cod liver oil, linseed oil, and a lotion. These were tested for stability at various temperatures. This preliminary study had a twofold purpose, (l) to verify the information that vanishing cream bases had a detrimental effect on vitamin A, and (2) to observe the effects of common ointment bases and similar substances on vitamin A. The ointment and liquid bases used were - 1. Hydrophilic ointment, U.S.P. 2. Lard 3. Vanishing cream (stearic acid and triethanolamine) 1|. Cod liver oil 5. Non ionic vanishing cream (stearic acid and tween) 6. Lotion buffered to pH = 5 (boric acid and tween) 7. Carbowax 8. Rose water ointment 9. Veegum HgO 10. Cholesterol base lanolin plus petrolatum 11. Vanishing cream with squalene (stearic acid and triethanolamine) 12. Linseed oil These samples were assayed for vitamin A by the U.S.P. XV method (97) and then samples of each were placed in 62 storage in an incubator kept at l\$° and others at room temperature. After months the former group kept at were reassayed for vitamin A. If there had been a large amount of decomposition the samples at room temperature were reassayed after 6 months. The results are given in Table 9. It appeared that the samples containing stearic acid (samples, 3, 6 and 11) exhibited greater losses than those where it was absent. Since there were several Ingredients in these ointment bases it would not be valid to state that the stearic acid was the causative agent. Since it was desirable to determine the nature of the changes occurring, the ointments showing the greatest losses were saponified with alcoholic KOH and the non saponifiable fraction was subjected to U.V. spectrum analysis. Two major types of reactions occurred: (l) oxi dation, evidenced by increased absorption below 326mu; (2) elimination, evidenced by increased absorption above 326mu. Since the preliminary study showed that there was considerable decomposition in ointment bases containing stearic acid this preliminary study was then extended to include the effect of hydrocarbon and alcoholic solutions of stearic acid on vitamin A. Vitamin A palmitate was dissolved in both a non polar solvent, n-hexane, and a polar bydroxylic solvent, n99$" isopropyl alcohol (water content = 0.056$ determined by the Karl Fisher method) (97) both containing 5 percent 63 TABLE 9 ASSAY VALUES FOR VITAMIN A IN VARIOUS OINTMENT BASES Initial ij. mo.l|5° 6 mo.R.T. % loss of Ointment base u/gm u/gm u/gm vitamin A 1. Hydrophilic oint. 10,300 7,090 31 2. Lard 8,000 6,800 15 3 •‘’“"Vanishing cream 111., 000 2,000 86 8,900 36 I4.. Cod liver oil 11,000 8,500 22.5 $.'rNon ionic 10,500 I4-H 96 vanishing cream 10,500 il-,700 55 b.^'Lotion buffered 11,000 l£l 96 to ph=5 (-h3bo3 ) 11,000 3,900 65 7. Carbowax 8,800 ij.,11.00 5 0 8. Rosewater oint. 9,11-00 5,500 1*1.5 9. Veegum/HgO 9,700 2,300 77 10. Cholesterol base 9,900 8,000 19 11 .'"Vanishing cream 8,500 1 ,5 0 0 83 with squalene 8,500 5,800 32 12. Linseed oil 13,000 9,500 27 ^Contains glycerine and stearic acid. stearic acid. All samples were examined for stability at 1+5° and room temperature and were assayed by the British Pharmacopeia method (96). The results are given in Table 10. TABLE 10 ASSAY VALUES FOR VITAMIN A IN n-HEXANE AND "99%" ISOPROPYL ALCOHOL Time in days 1199%" Isopropyl alcohol n-Hexane o • Room temp, £ 1 Room temp, 1+5° u/ml u/ml u/ml 0 10,950 10,950 10,950 10,950 9,709 8,778 9,81+2 9,709 8 9, 1+1+3 7,81+7 9,709 9,576 15 9,053 6,111 9,1+62 9,557 19 9,1+22 3,322 9,319 9,699 35 9,032 70 9,566 1,997* ^Cap fell off and oxidation set in. In order to determine the nature of changes occurring in these samples, periodic U.V. examinations were made. The changes occurring within 19 days are summarized as follows: 1. n-Hexane (a) No anhydrovitamin A formed. (b) Slight degree of oxidation. 6 5 2. "99% " Isopropyl alcohol (a) Definite quantities of anhydrovitamin A formed. (b) Slight degree of oxidation. When the reaction was allowed to proceed for 35 days, the changes were more pronounced. Oxidation was very marked in n-hexane at 45° and less at room temperature. The sample in if99%" isopropyl alcohol at 1^5° showed complete loss of vitamin A with maxima in the region of oxidation products; small amounts of anhydrovitamin A still remained. The samples at room temperature demonstrated mostly elimination products. A plot of log concentration vs. time of the isopropyl alcohol sample at L|50 was a straight line, indicating a first order dependence on the concentration of vitamin A. The first order rate constant was 1.26 x 10”^hr“^-. It can be inferred from these data that no anhydro vitamin A was formed in hydrocarbon solutions, but was formed in alcoholic solvents. These samples were chromatographed on slightly deactivated alumina to determine: 1. The nature of the decomposition products. 2. The best type of alumina for chromatographing these samples. If the alumina was too strong, it caused changes in some of the constituents; if too weak, separation was not satisfactory. 66 The isopropyl alcohol samples at 1 $ ° were selected for chromatography because these samples showed the greatest variety of change and would probably represent the greatest differential in number of ingredients that could be encountered. The alumina was deactivated by adding 3, 6, and 10$ of water to anhydrous activated neutral alumina. The details of the chromatography will be discussed in a later section. The results are summarized as follows: 1. Type I alumina (no water added) was not suitable since it caused the formation of artefacts as red pigments and more anhydrovitamin A than was originally present. 2. Type II (3$ water) also appeared to be too strong. 3« Type III (6$ water) was satisfactory. ij.. Type IV (10$ water) was too weak. When these samples were chromatographed on type III alumina the components of the mixture were eluted in the following order: anhydrovitamin A, vitamin A palmitate, a substance with a maximum at 228mu (oxidation product) and substances with maxima at 270— > 290mu (oxidation products). The isopropyl alcohol sample at l\S° contained relatively little anhydrovitamin A but large amounts of oxidation products (maxima 270-286mu). The room temperature sample contained less oxidation products. During this preliminary chromatographic experiment, it was noted that the method of chromatography could be 67 improved by proper use of the polar solvents. When these solvents were added too quickly poor zones of separation resulted. This information was utilized in later work. DETERMINATION OP THE STABILITY OP VITAMIN A IN THE PRESENCE OP AN HOMOLOGOUS SERIES OP PATTY ACIDS Reagents Used in This Study 1. Fischer Reagent grade Stearic Acid MP-b8°-69.5° 2. Fischer Laboratory Chemical Oleic Acid (Purified) 3. Fischer Laboratory Chemical Caproic Acid (Purified) !}.. Pischer Reagent Chemical Palmitic Acid MP-61°-62° 5. Eastman Kodak Decanoic Acid b. Dupont Reagent Glacial AceticAcid 99*7$ 7. Sohio "99$” Isopropyl Alcohol (0.056$ water) 8. USP Ethyl Alcohol 9. Baker Anhydrous Acetone 10. Mallinckrodt Anhydrous Ethyl Ether 11. Whatman filter paper #1 (l-g-”) for chromatography 12. Heavy liquid petrolatum USP 13. Baker’s Light Petroleum Ether BP-*30o-60° (AR) li^. Cyclohexane Phillips Petroleum Products (redistilled) 15* n-Hexane (technical) OSU label (redistilled) 16. Woelm Neutral Activated Alumina for Chromatography (M. Woelm, Eschwage, Germany) 17* Mallinckrodt Absolute Methanol 18. Vitamin A Palmitate, Type (PIMO), Hoffmann La Roche, Inc. 19. Vitamin A Acetate, 2,900,000 U/gm, Hoffman La Roche, Inc. 20. Butylated Hydroxy Toluene (BHT), Pood Grade (Koppers Co., Inc., Pittsburgh, Pennsylvania. 6 8 Decomposition Rates of Esters In "99$"'* Isopropyl Alcohol In this study it was desired to determine whether (1) fatty acids would accelerate anhydrovitamin A formation when compared to a control containing no fatty acid; (2 ) there was any difference in rates of anhydrovitamin A formation produced by caproic, decanoic, palmitic, oleic, or stearic acids; (3 ) decomposition other than elimination occurred and if so, what was the nature of this loss. In order to determine the rates of vitamin A de composition, and order of dependence on vitamin A, It was thought desirable to have all the other ingredients in large excess of the concentration of vitamin A. The solutions were made in "99%" isopropyl alcohol with an approximate concentration of 10,000 units of vitamin A palmitate per ml. The fatty acids were used as N/30 solutions. This concentration was chosen because N/3 0 mineral acids were known to have a destructive action on vitamin A. It was also desired to examine the difference in effects of these two types of acids. The choice of isopropyl alcohol as the solvent throughout these experiments was based upon several con siderations. The solvent should be water miscible, good solvent for vitamin A and fatty acids, and having only slight activity upon vitamin A. Chilcote et al. (90) showed ^Labelled "$9%" Isopropyl Alcohol; a Karl Pisher determination showed 0 .056% water content. 69 in the homologous series of alcohols, that methanol was^ Ethanol ^ Isopropanol in their destructive ability upon vitamin A. The reason for this might be explained by Hine and Hine (157) who demonstrated the relative acidities of a large number of alcohols and acids. Based on an arbitrary standard, methanol had an acidity Lj..0 0 ; ethanol 0 .9 5? water 1 .00; and isopropyl alcohol 0 .076. Preparation of the Solutions The following method was used for all the experiments. The "99%" isopropyl alcohol was flushed with nitrogen for one hour. Quantities of caproic, decanoic, palmitic, oleic, and stearic acids were dissolved in this solvent to make N/30 solutions; vitamin A palmitate was added to make an approximate concentration of 10,000 U/ml. A control of vitamin A palmitate in ”99%" isopropyl alcohol without fatty acid was used* These solutions were placed in 120 ml glass stoppered amber bottles and immediately assayed by the British Pharmacopeia method (96). The containers were flushed with nitrogen and stored at I+50, 37°, and 25°. Each time a container was opened to withdraw a sample it was re- flushed with nitrogen. No fatty acid was found to be lost due to esterification with the alcohol, as determined by titration with base. None of the fatty acids exhibited any appreciable U.V. absorption in the region of vitamin A; thus it was 70 shown that the British Pharmacopeia Method of vitamin A assay was applicable without interference from the fatty acids. A plot of the degradation of vitamin A followed a logarithmic law and was shown to be first order with respect to vitamin A. This experiment was performed over a period of 650 hours. The rates are shown in Table 11, expressed as the rate k x 10^ per hour (k x 10^ hr."1 ). TABLE 11 RATE CONSTANTS FOR DECS? AD ATI ON OF VITAMIN A IN "99%" ISOPROPYL ALCOHOL SOLUTION (k x hr."1 x 10^) "99%" Isopropyl N/30 N/3 0 N/30 N/30 N/30 Alcohol Caproic Decanoic Palmitic Oleic Stearic Control Acid Acid Acid Acid Acid l|5°- k-^ 13.7 1 2 .0 1 2 .J? 11^.9 15-0 1 5 .0 k5°- k2 1 .5 0 1 .0 0 1.13 1 .05 1.33 1.25 37°- kl 1 2 .0 7.70 5.30 5-90 7.01 8.00 37°- k2 1.25 0.980 0.938 0.833 0.910 0.831*- <1 LOO CONCENTRATION OF VITAHI* A ( U/ol.) U/ol.) A ( VITAHI* OF CONCENTRATION LOO U/ml.) A ( VliAMIN OF CONCENTRATION LOO TIME : HCUBS FI3URK 2 72 Since this series demonstrated only quantitative differences, the graph of vitamin A palmitate in "99%n isopropyl alcohol, Figure 1, is submitted as representative of the entire group. An examination of this graph and of Table 11 shows the following data: 1. At I4.50 and 37°, each graph appears to be composed of two different rates, labeled as k-^ and kg in Table 11. 2. The k$° sample shows an unexpected drop in the region of 300 hours• 3. The 25° sample showed an initial drop, a rise and then another drop without a subsequent rise. i).. The rate constants, except for a few cases (k-^ for stearic, palmitic, and oleic acids at only), indicate that the rate of destruction of vitamin A in the presence of fatty acids, is less than in the control which contains no fatty acid. 5. The graph indicates a pseudo first order reaction depending only on the concentration of vitamin A. The pH was measured in order to correlate the rates of decomposition with the pH of the solutions. The con ventional pH meter was unsatisfactory because it is designed to measure pH of aqueous solutions. Using the La Motte Color Indicators the fatty acid solutions had a pH of approximately 5.5 and the pH of "99% " isopropyl alcohol was 5.7. It was evident that these were relative pH values and not absolute. 7 3 The U.V. spectra were determined periodically and were found to possess the absorption spectrum of vitamin A which insured that the assay method was still valid. These spectral analyses demonstrated by virtue of the change in maxima that the two major decomposition products were anhydro vitamin A (elimination) and vitamin A epoxide (oxidation); the amounts of each increasing with the temperature. The positive identification of these products was not possible in this mixture because the spectrum of the components of the solution interfered with each other. Consequently the solutions were chromatographed on alumina in order to isolate the individual components. The adsorbent used was Type III Woelm alumina pre pared by deactivating the anhydrous alumina x*jlth 6% of water. After 2 hours of hydration it was poured into n-hexane to be used as a slurry. A glass column 10" long and 0.75" wide was filled with alumina from the slurry and allowed to settle. The reaction mixture containing the fatty acid, vitamin A and decomposition products was dried in vacuo at room temperature, redissolved in 5 ml n-hexane and poured on the alumina column. The eluant was added immediately after the sample, without allowing the column to become dry. The weakest eluant, n-hexane, was used first and each fraction measured 25 ml. Anhydrovitamin A was the first compound eluted. The vitamin A esters which followed anhydrovitamin A down the column were also removed by n-hexane. After the ester and hydrocarbon had been removed, the next fractions of eluate had very weak U.V. absorption. At this point it was necessary to use a stronger eluant; 2 percent ethyl ether in n-hexane was used. The transition point was designated as that point where the eluant could no longer remove material from the column. At each transition point it was necessary to increase the strength of the eluant in the following fashion: i|., 8, 16, 25> i|0, 5 0, and 75 percent ethyl ether in n-hexane. The choice of eluants required considerable care since these solvents should not react with the ingredients and should give good separation. Acetone could not be used because it has considerable U.V. absorption in the region of 300mu and consequently interfered with the assay for vitamin A. The U.V. spectrum of each fraction was determined on a Beckman DK Recording Spectrophotometer, then the solvent was removed in vacuo and the I.R. spectrum was determined with a Baird Infrared Spectrophotometer. A compilation of these results is found in Table 12. The components of the mixture were eluted from the column in the following order: 1. Anhydrovitamin A 2. Vitamin A ester 3. Esterified oxidation product I4.. Rehydrovitamin A 5. Substance with maximum at 285mu (oxidation products) 6 . Substance with maximum at 280mu and a stronger maximum at 235mu (oxidation product) 7 5 TABLE 12 U.V. AND I.R. SPECTRA OP CHROMATOGRAPHED FRACTIONS ("99%" ISOPROPYL ALCOHOL AND N/30 PATTY ACIDS) Sample-N/30 Caproic Acid-n99$" Isopropyl Alcohol 1+5° Groups demonstrated Fraction No U.V.Spectrum by I. R. Spectrum 1 - 2 anhydrovitamin A hydrocarbon 3 - 12 vitamin A substance ester (max. 326mu) 13 - 15 oxidation products hydr oxy1-ester (max. 281, 291mu) 16 - 18 oxidation product hydroxyl (max. 285m.u) 19 - 20 330, 3i+6, 36ipiu 21 - 23 max. 286mu hydroxyl-ester 21+ - 31 max. 28lmu, below 235mu strong hydroxyl 2. Sample-N/3 0 Stearic Acid-n99%" Isopropyl Alcohol l+£° 1 - 3 anhydrovitamin A hydrocarbon k - 12 vitamin A substance ester max. 326 13 - 15 oxidation product hydroxyl-ester max. 282 16 - 17 max. 381+, 3 M 5, 330mu 18 - 20 oxidation product hydroxyl-ester 25 - 29 max. 287mu hydroxyl 30 - 31 max. 331* 31+8, 361+mu 33 - 37 max. 282mu & below 235mu hydroxyl TABLE 12-(Contd.) 3 . Sample-'^^" Isopropyl Alcohol Control I4.5 0 Fraction No. U.V.Spectrum Groups demonstrated by I.R. Spectrum 1 - 6 anhydrovitamin A hydrocarbon 7 - ib vitamin A substance ester and ether 18 - 19 max. at 280mu ester and hydroxyl 20 - 2b max. 365, 314-7, 330mu 27 - 29 max. 287mu hydroxyl 30 - 37 max. 285, 290mu hydroxyl 14- Sample N/30-Palmitic Acid in n99%" Isopropyl Alcohol bS° 1 - 2 anhydrovitamin A hydrocarbon 3 - 9 vitamin A substance ester - ether max. 326mu 15 - 16 280mu hydroxyl-ester (oxidation products) 0 f~- CVJ 1—1 1 326-vitamin A alcohol hydroxyl-ester 295, 280 - oxidation, products 21 - 23 332, 314-7 and 367mu hydroxyl 28 - 29 max. 29 Omu hydroxyl 33 - 39 max. 285mu & below 235rau hydroxyl 77 TABLE 12-(Contd.) 5. Sample N/30-01eic Acid in "99%" Isopropyl Alcohol 1+5° Groups demonstrated Fraction No. U.V.Spectrum by I.R. Spectrum 1 - 2 anhydrovitamin A hydrocarbon 3 - 9 vitamin A substance ester (max. 326mu) 11 - 11+ max. 28lmu e s ter-hydroxyl 16 - 18 max. 28lmu ester-hydroxyl 19 - 21 max. 32^ hydroxyl (vitamin A alcohol) 23 - 21+ 330, 3k$> 36£mu 25 - 26 max. 28 6 ester-hydroxyl 27 - 31 max. 285 hydroxyl 32 - 31+ max. 285 & below 325mu hydroxyl 6. Sample-"99%" Isopropyl Alcohol Control 25° 1 - 2 anhydrovitamin A hydrocarbon 3 - 12 vitamin A substance ester (max. 326mu) 16 17 max. 282mu ester-hydroxyl 18 max. 3^7» 34-8, 3 3 3 ^ 19 - 22 max. 285mu hydroxyl 23 - 29 max. 285 & below 23!?mu hydroxyl 78 TABLE 12-(Contd.) 7 # Sample N/30-Palmitic Acid in ”99%" Isopropyl Alcohol 25° Groups demonstrated Fraction No. TJ.V. Spectrum by I.R. Spectrum 1 - anhydrovitamin A hydrocarbon 2 - 9 vitamin A substance ester max. 326 10 - 15 max. 280mu ester-hydroxyl 18 - 21 max. 285mu ester-hydroxyl 22 - Zk 367, 3k9> 333mu hydroxyl 25 - 27 max. 285mu hydroxyl 28 - 33 max. 285mu & below 235niu hydroxyl 8. Sample N/30-Decanoic Acid in ,,99%" Isopropyl Alcohol 25° 1 - 0 anhydrovitamin A hydrocarbon 3 - 10 vitamin A substance ester max. 326 12 - 13 max. 280mu hydroxyl-ester 15 - 16 max. 33O, 3I4.9, 369mu 18 - 27 max. 280mu hydr oxyl-e s t er 28 - 31 max. 288mu hydroxyl-ester 33 - 3k max. 285mu hydroxyl-ester 37 - 39 max. 285 & below 235*au hydroxyl 79 Table 12-(Contd.) 9. Sample N/30-Caproic Acid in "99%" Isopropyl Alcohol 25>° Groups demonstrated Fraction No. U.V.Spectrum by I.R. Spectrum 1 anhydrovitamin A hydrocarbon 2 - 9 vitamin A substance ester (max. 326) 15 - lb max. 280mu ester-hydroxyl 19 - 23 max. 28lmu ester-hydroxyl 26 - 28 max. 29 Omu hydroxyl 31+ 333, 31+7, 367mu 35 - 36 max.282mu & below 235mu hydroxyl 10. Sample N/30-01eic Acid in "99%" Isopropyl Alcohol 2$° 1 - 2 anhydrovitamin A hydrocarbon 3-13 vitamin A substance ester (max. 326) 16 - 19 max. 282 ester-hydroxyl 20 max. 327, 312, 29l+mu ester-hydroxyl 22 - 2k max. 285diu ester-hydroxyl 25 - 29 max. 285 & below 2 3 5 ^ hydroxyl 80 Addition of Water to ”99%" Isopropyl Alcohol Samples Since the presence of fatty acids in the "99%" isopropyl alcohol samples decreased vitamin A loss^5 percent of water was added to the media in order to observe the effects of possible increased ionization of the fatty acids. It was also desired to determine the effect of water on this system. A quantity of distilled water was added to the "99%" isopropyl alcohol to make a "95$" isopropyl alcohol solution. A Karl Fisher tirtration (97) showed that the actual content of water was L|..66 percent. This same solvent was used in all further experiments calling for "95%" isopropyl alcohol and will be referred to as "95%" isopropyl alcohol. The solvent was flushed with nitrogen for one hour and quantities of caproic, decanoic, palmitic, oleic and stearic acids were added to make N/30 solutions. Due to the epiphasic nature of vitamin A palmitate, concentrations equivalent to 10,000 TJ/ml could not be attained in this solvent. Consequently it was found necessary to replace vitamin A palmitate with vitamin A acetate (Roche 2.9 M U/gm). The samples were prepared and treated in the same manner as the "99%" isopropyl alcohol samples. They were stored at 25°, 37°, and lj-50. Indicators were used to determine the pH of these solutions. The addition of I4..66 percent water caused a slight decrease in the relative pH. N/3 0 fatty acid solutions in "95%" isopropyl alcohol had a pH = I4..I4. and the 81 ”95%" Isopropyl alcohol had a pH = 5*8. A new method of assay for vitamin A (128) which used isopropyl alcohol as the diluting solvent was adopted because the water present was not miscible with cyclohexane on the B.P. method. The method of calculation is as follows: — ^ Eg — 326 mu, ^ E^ = 338mu = 3.61 (2Ep - E. - E ) lcm (corr.) Z l 3 Percent dilution x 1 Potency in TJ/ml = E1^ x 1900 lcm, corr. ^ = length of cell in cm The testing period was allowed to proceed for 120 hours because at that time the b£° samples exhibited approximately 60 percent loss. This time was considerably less than that required for the "99%" isopropyl alcohol samples to reach this loss. It was evident that water exerts some catalytic effect on this reaction. The presence of the fatty acid again showed two distinct effects: 1. Decreased vitamin A loss. 2. Decreased the amount of anhydrovitamin A in solution. The log of the concentration of vitamin A versus time was plotted. The results fell on a straight line indicating a psuedo first order reaction. Since these plots demon strated quantitative differences, only the plot of the 82 control vitamin A acetate in isopropyl alcohol is shown in Figure 2, and Table 13 gives the rates of decomposition. TABLE 13 RATE CONSTANTS FOR THE DEGRADATION OF VITAMIN A IN "95%" ISOPROPYL ALCOHOL k x (hr“l x lO^) 1+5° 37° 25° Control (no fatty acid) 1+.17 1*1+3 0.39 N/30 Caproic acid 2.35 0.77 0.21+ N/30 Decanoic acid 2.1+5 1.01 0.1+1 N/30 Palmitic acid 2.1+0 0.83 0.26 N/30 Oleic acid 2.50 0.72 0.27 N/30 Stearic acid 2.79 1.00 0.56 Five percent water accelerated the rates of decomposition in all cases. The fatty acid samples showed slower rates when compared to a control containing no fatty acid. Since vitamin A exhibits at 391mu approximately 1 percent of the extinction at 326mu, and anhydrovitamin A has a maximum at 391mu = 3180, the extinction at 391mu would be a good quantitative measure of the rate of anhydrovitamin A formation. It was desired to correlate the amount of anhydrovitamin A formed with the corresponding units vitamin A lost. The following formula was derived to 8 3 convert into the equivalent units of vitamin A: 391 x 1115?______Units of vitamin A converted l(cm) x percent dilution into anhydrovitamin A A plot of the log (A^ - A^.) vs t- where A^ repre sented the original -units of vitamin A per ml and A^. the units of vitamin A converted into anhydrovitamin, was a straight line. She slope of this line gave the rate of conversion of vitamin A into anhydrovitamin A. The rates are given in Tableli|. and the graph of the 45° samples is given in Figure 3* It appears that at least one-half of the decomposition products is anhydrovitamin A. TABLE II4 INITIAL RATES OF FORMATION OF ANHYDROVITAMIN A IN UNITS OF VITAMIN A ACETATE LOST IN "9S%" ISOPROPYL ALCOHOL k x (hr“l x 103) k5° 37° Control (no fatty acid) 2.0 0 0.528 N/30 Caproic acid 1 .1I4 0 .1|2l|. N/3 0 Decanoic acid 1 .1 0 O.lp-5 N/30 Palmitic acid 1 .2 0 0.399 N/3 0 Oleic acid 1 .32 0 .14.324. N/3 0 Stearic acid l.o^ 0.3824 81+ It was also desired to compare the total amount of vitamin A lost with that converted into anhydrovitamin A at 110-111+ hours by the formula on page 8 3. These results are given in Table 15. TABLE 15 COMPARISON OP TOTAL VITAMIN A LOSS BY CONVERSION INTO ANHYDROVITAMIN A IN ”95$" ISOPROPYL ALCOHOL Ho '0 N/3 0 N/3 0 N/3 0 N/3 0 N/3 0 Isopropyl Caproic Decanoic Palmitic Oleic Stearic Alcohol Acid Acid Acid Acid Acid ______Control______ 1+5° - Total loss 5ll+9 1+221 1+310 1+033 3923 3895 Vitamin A-> Anhydro- 2732 2107 2007 2263 2196 2000 vitamin A 37° - Total loss 251+0 1823 191+2 11+21+ 1525 1367 Vitamin A-^ Anhydro- 11+16 959 911+ 937 937 903 vitamin A 25° - Total loss 31+3 281). 232 326 291 286 Vitamin A-^ Anhydro- 267 223 223 23I+ 2l+5 223 vitamin A 8 & CONVERSION OF VITAMIN A ACSTAIZ i:.TQ •yracvirAXi.N A IK " 95< " isc?ac?^L a l c c h c : 5S 3 < ■< o 3 100 "HHCMATOjRAPHY CF M/^C ?AL.’g 25£ " ISCm cpg. ALCOHOL f=° SAUFtS < ^ = i nMn»m3Sl.3M 9W-m < Oj5:1 Wfl FRACTION :r.'M3EK 8 b CHROMATOGRAPH! OF jlAO CAPROIC ACID " ?5< " ISOfROiTL ALCOHOL 5AXgLE 4-5 9 m PRAOTICS SCTfflg- n s c a 5 CHRomatoorapht or * 95< * laoraofn. alcohol control sample ^.5* m IOC i ,0= MAXIMUM-. 3t8n»u. oW <25 a Ctl oa tfio 40 00 15 FRACTION NUHBSR FI3URK 6 8 7 An examination of Table If? shows that the fatty acids decreased the amount of degradation into anhydrovitamin A, consequently it was desired to determine the other methods of decomposition by chromatographic analysis. Oxidation was found to be the other major decomposition pathway. The N/3 0 palmitic acid, N/3 0 caproic acid and con trol samples in "95%" isopropyl alcohol at I4.50 were selected for chromatographic analysis. They were dried in vacuo and promptly dissolved in n-hexane and chromatographed on Woelm Alumina columns deactivated by the addition of £ percent water. The solvents and procedure were the same as in the chromatography of the "99%" isopropyl alcohol series. Since many of the fractions of eluted material con tained identical substances, it was not necessary to subject each to U.V. and I.R. spectroscopy. By plotting U.V. ex tinction versus fraction number new substance being eluted from the column could be readily detected. The initial fractions had a low concentration of eluate. The concen tration rose in the subsequent fractions until a maximum was reached represented by the highest peak on the graph. The concentration of each subsequent fraction decreased until no more was eluted. Several of the following fractions may not contain any eluate; if a stronger eluant was added, a new substance came off and the above pattern was repeated. This pattern was found with the three chromatographed samples. The above-mentioned graphs are shown in Figures I4.-6 . The center fractions were the most concentrated and were 88 taken as representative of the particular group. This fraction was subjected to U.V. and I.R. spectroscopy. The results of the spectroscopic analyses are presented in Table 16. It is obvious that considerable oxidation occurred along with elimination. Thus the presence of an antioxidant in preparations containing vitamin A is necessary. TABLE 16 U.V. AND I.R. SPECTRA OP ”95$" ISOFROPYL ALCOHOL - VITAMIN A ACETATE CONTROL i|5° Fraction Groups demonstrated Number U.V.Spectra by I.R. Spectra 3 max. at 351, 371, 391mu unsaturated anhydrovitamin A hydrocarbon 7 max. at 328mu, vitamin A ether 15 max. at 328mu, vitamin A ester 21+ max. at 368, 31+8, 328, 311+mu hydroxyl-ester mixture of isoanhydrovitamin A and oxidation products 30 max. at 312, 295mu hy dr oxyl - e s t er 38 max. at 326mu,vit.A alcohol strong hydroxyl 1+3 max. at 285-290mu hydroxyl oxidation product 89 TABLE 16-(Contd.) U.V. AND I.R. SPECTRA OP "95%11 ISOPROPYL ALCOHOL - VITAMIN A ACETATE, N/30 PALMITIC ACID 45° Fraction Groups demonstrated Number U.V.Spectra by I.R. Spectra 2 max. at 391, 371, 351niu unsaturated anhydrovitamin A hydrocarbon 8 max. at 326mu(vit.A substance) ester 27 max. at 388, 3l|.6 , 325, 312, hydroxyl-ester 297mu, esterified oxidation products & isoanhydrovitamin A 36 max. at 326 mu, inflection hydroxyl at 311» 295rau, vitamin A alcohol, oxidation products max. at 285-290, 235mu, hydroxyl unesterified oxidation products U.V. AND I.R. SPECTRA OP "95%" ISOPROPYL ALCOHOL - VITAMIN A ACETATE N/3 0 CAPROIC ACID ij.50 1 max. at 391, 371, 35lmu unsaturated hydrocarbon k max. at 326 mu, ester vitamin A substance 11 max. at 326, 311» 296, 282mu hydroxyl-ester esterified oxidation products 15 max. at 325mu hydroxyl vitamin A alcohol 20 max. at 371, 351, 332mu 29 max. at 288-290mu hydroxyl oxidation product 9 0 Each of the representative fractions designated in Table 16 were paper chromatographed in order to determine if they were mixtures or single substances. The appearance of one zone indicated a single substance unless two or more substances having identical Rf values were present, which is unlikely. In all cases Whatman #1 (l-g-") strip filter paper was used. The stationary phase consisted of a lipophilic material impregnated into the paper, by applying a 5 percent solution in petroleum ether (30° - 60°) and allowing the solvent to evaporate. A spot of the material to be analyzed was put on the starting line and developed with the pre scribed solvent in a large glass tank. There were eleven combinations of (lipophilic material - eluants) tried: 1. Heavy liquid petrolatum 5 percent - Ethanol USP 2. Heavy liquid petrolatum 5 percent - 50$ Isopropyl alcohol/water 3. Olive oil 5 percent - Ethanol USP 1|. Heavy liquid petrolatum 5 percent - 50$ Isopropyl alcohol/water 5. Heavy liquid petrolatum 5 percent - Methanol/ ethanol USP 6. Olive oil 5 percent - Methanol/ethanol USP 7. Heavy liquid petrolatum 5 percent - 90$ Isopropyl alcohol/water b. Olive oil 5 percent - 90$ Isopropyl alcohol/ water 91 9. Coconut oil 5 percent - Methanol/ethanol USP 10. Sesame oil 5 percent - 90% Isopropyl alcohol/ water 11. Sesame oil 5 percent - Methanol/ethanol USP When the "95%" isopropyl alcohol control sample at L\.S° was chromatographed with the above mentioned systems, the 5 per cent olive oil impregnated paper with a methanol-ethanol eluant appeared to be the best. The results are tabulated in Table 17* TABLE 17 Rf VALUES OP CHROMATOGRAPHED "95%" ISOPROPYL ALCOHOL CONTROL lg° Fraction Number or Substance Rf Values Possible Identification Vitamin A acetate 0.1)35 Vitamin A acetate ( c o n t r o l ) Vitamin A alcohol 0.777 Vitamin A alcohol ( c o n t r o l ) 7 0.393 Vitamin A isopropyl ether 15 0.1p58 Vitamin A acetate 31 0.760 Vitamin A alcohol 37 0.75^/0.836 Vitamin A alcohol- oxidation product k3 0.81)7 Oxidation product 92 The fractions from the sample containing vitamin A acetate in N/30 caproic acid - "95%" isopropyl alcohol at 4-5°, responded best to a system consisting of paper impregnated with 5 percent heavy liquid petrolatum and eluted with 50 percent methanol-ethanol. These results are in Table 18. TABLE 18 Rf VALUES OP CHROMA TO GRAPHED "95%" ISOPROPYL ALCOHOL - N/30 CAPROIC ACID l\$° Fraction Number Rf Values Possible Identification Vitamin A acetate 0.519 Vitamin A acetate (control) Vitamin A ale ohol 0.858 Vitamin A alcohol (control) 4 0.300/0.54.7 Vitamin A ester/vitamin A acetate 11 O.488/O. 633 / Esterified oxidation produc O .847 and latter is vitamin A alcohol 15 0.871 Vitamin A alcohol 20 29 0.895 Unesterified oxidation product The fractions from the sample containing vitamin A acetate in N/3 0 palmitic acid - "95%" isopropyl alcohol 45° responded best to a system consisting of filter paper 9 3 impregnated with 5 percent heavy liquid petrolatum using ethanol as the eluant. Results are found in Table 19* TABLE 19 Rf VALUES OP CHROMATOGRAPHED "95%" ISOPROPYL ALC0H0L-N/30 PALMITIC ACID l±$° Fraction Humber Rf Values Possible Identification Vitamin A acetate 0.514 Vitamin A acetate Vitamin A alcohol 0.895 Vitamin A alcohol 8 0.057/0.520 Vitamin A palmitate/ vitamin A acetate 27 O.38I4./O.8O5 Esterified oxidation products 35 0 . 1^ 7/ 0.900 Vitamin A alcohol k2 0.951 Unesterified oxidation product In this series of experiments the possible identifi cation is based on the integration of the U.V. spectrum, I.R. spectrum, Rf values, color, solubility in alcohol and in petroleum ether. In certain cases supposedly individual fractions were found to consist of mixtures. This is a good indication that their separation on the alumina columns entails a degree of uncertainty. In the three cases studied here the vitamin A active fraction separated into at least two or more fractions. This has some significance since only one vitamin A active 91*. substance (vitamin A acetate) was originally introduced. This indicated that there was interconversion of vitamin A active forms, and was clearly demonstrated when the vitamin A active fraction (No. 7) of the palmitic acid - "9$%" isopropyl alcohol h$° sample was chromatographed on paper impregnated with 6 percent liquid petrolatum using a methanol- ethanol eluant. It separated into three fractions: 1. Rf 0.082;-Yellow in U.V. light; U.V. spectrum of vitamin A corresponding to vitamin A palmitate. 2. Rf 0.5315-Yellow in U.V. light; U.V. spectrum of vitamin A corresponding to vitamin A isopropyl ether. 3 . Rf O.b71;-Yellow in U.V. light; U.V. spectrum of vitamin A corresponding to vitamin A acetate. The ratio of these substances is 1:3*5:25 The 7th and l5th fractions of the "95%” isopropyl alcohol control L\.$° showed different Rf values and similar U.V. spectra indicating that they are different substances. Fraction No. 7 showed an I.R. spectrum corresponding to an ether with no ester and could be concluded to be vitamin A isopropyl ether. Fraction No. 15 showed an ester group and could be assumed to be vitamin A acetate. Fraction No. (vitamin A active fraction) of the sample containing N/3 0 caproic acid I\$° was found to be a mixture of vitamin A active substances. One of these was thought to be vitamin A acetate on the basis of U.V. and I.R. spectra and Rf value when compared to the control. There was another substance present that consistently had a 95 lower Rf value than vitamin A acetate, was yellow in U.V. light and had the U.V. spectrum of vitamin A and was believed to be the caproate. It was unlikely that the unidentified material was vitamin A ether since the Rf values did not compare favorably. Since the other fractions also separated into various parts, it indicated that they were either mixtures or that some change had occurred on the paper during chromo- tography. Since the spots were well defined it ruled out the latter possibility. This separation was found in fractions containing oxidation products. It could be concluded that a number of oxidation products with similar structures were formed in the systems studied. These oxidation products showed Rf values in the region of 0.895 to 0.950, depending on the system. It appeared that paper chromatography was a good method of qualitatively determining If oxidation had occurred in a vitamin A ester sample. The Rf values of the oxidized material were slightly higher on liquid petrolatum im pregnated paper than on paper impregnated with fixed oils. This is logical since the oxidation products are less soluble In petrolatum than in fixed oils. These systems contained the following substances: 1. Vitamin A acetate 2. Vitamin A ester formed from fatty acid of media 3. Vitamin A alcohol 96 i|. Possibly vitamin A ether 5. Oxidation products 6 . Anhydrovitamin A ACTION OP ISOPROPYL ALCOHOL, WATER, CYCLOHEXANE AND PATTY ACIDS ON ANHYDROVITAMIN A The previous experiments indicated that fatty acids decreased the amount of anhydrovitamin A and simultaneously maintained a higher level of vitamin A. Apparently the fatty acid stabilized the solutions by a catalytic regener ation of a vitamin A substance (ether, ester or alcohol). Consequently, it was Delieved that a study of the action of water, isopropyl alcohol, and fatty acids on anhydrovitamin A would lead to some valuable information regarding the nature of this stabilization. Since the previous part of this study showed that differences in stability could be expected when vitamin A was dissolved in alcoholic, hydroalcoholic and non polar hydrocarbon solvents, it was decided to study the action of fatty acids in anhydrovitamin A in the types of solvents mentioned. The solvents used in this study were cyclohexane, n99% n isopropyl alcohol (0 .0^6$ water), 1195%11 isopropyl alcohol (L\.,bbfo water), and an isopropyl alcohol having water content of II4..36^ as determined by a Karl Pisher titration (97)• All these solvents were flushed with dry nitrogen for two hours prior to their use. 97 To insure the correct measurement of vitamin A substance formed, it was necessary to prevent the loss through oxidation; consequently, 0.1 percent BHT (butylated hydroxy toluene) was added. As a further means of prevent ing oxidation, the bottles were flushed with nitrogen every time they were opened to remove a sample. An examination of the spectrum of the BHT showed that it would not interfere with the spectroscopic analysis of vitamin A. Cyclohexane was purified by distillation and chroma tographing on a completely activated alumina. The purified solvent was transparent above 2i|.Qmu. The isopropyl alcohol was purified by distillation between 81° - 83°, the boiling point range prescribed by the USP (97). The anhydrovitamin A was prepared according to the method of Shantz (lijl). The reaction mixture was chromato graphed on Woelm alumina, deactivated by the addition of 3 percent water, according to the method previously described. Anhydrovitamin A was the first material to be eluted from the column. It was rechromatographed on second column to effect better purification. Shantz (II4.I) determined the degree of purity of the anhydrovitamin A by the ratio of the extinction in ethanol at 391mu/371mu = O .87 and 35lmu/37lmu = 0.68. The material conformed to these standards. A system was devised to calculate the amounts of vitamin A and anhydrovitamin A present at any time. This 98 problem was complicated by the fact that their U.V. ab sorption spectra overlapped and direct extinctions could not be used to calculate the amount of each substance present. Using the following information, simultaneous equations were derived: Anhydrovitamin A: E }^° x = 2500* = 700; A„, = 0.290 lcm (35imu) lcm (32bmu) A351 Vitamin A alcohol: Blcm (3Sl»m) = 9°3’ Eicm (320mu) = l8» : “ 2'°32 A35i Let x = absorption due to anhydrovitamin A at 35lmu Let y = absorption due to vitamin A at 351mu 1. x + y = E3t>1 2. 0.290x + 2.032y = E326 3. x = 1 .lb6 - 0.571+ E^g^ 1+. y = 0.571+ ^326 *" 9*1^8 E35^ In this first series only two fatty acids were tested using the cyclohexane as the solvent. It was believed that the pertinent information concerning the reaction could be obtained by the use of acetic and caproic acids. The solutions were made up to contain 2, 1+, and 6 molar acetic acid and again using caproic acids in cyclohexane, containing 0.1 percent BHT. The anhydrovitamin A concentrate was added to these solutions, giving approximately 0.1 percent solution or (0.0037M). Each of these samples was divided 99 into three parts and stored at 25°, 37°, and 50°. A control was used, containing the anhydrovitamin A plus 0.1 percent BHT in cyclohexane and was treated exactly like the other samples. The samples were assayed by diluting 1 ml of the original material with 99 ml of isopropyl alcohol. The extinctions of these dilutions at 326, 351* 371* and 391mu were measured on a Beckman DU spectrophotometer. Formulas 3 and ij. above were used to calculate the extinctions of vitamin A and anhydrovitamin A at 351mu. The extinctions at 371 and 391mu were desirable because they gave information regarding changes in the nature of the reactants. The extinction due to vitamin A at 391mu is negligible. The examination of the extinction at these wavelengths could show if any secondary reactions were occurring which might interfere with the determinations at 326 and 35lmu. Periodically, the absorptions at 280mu were determined in order to ascertain if any oxidation was occurring. These experiments were designed so that the fatty acids would be in a large excess of the concentration of the substrate*, consequently the order of dependence on anhydro vitamin A could be determined from a suitable plot of the data (58). The solutions containing 0.1 percent anhydro vitamin A (0.0037M) were satisfactory because the fatty acids and solvent (cyclohexane) would be in large excess and would not enter into the rate equation. If the log C versus 1 0 0 t were shown to be a straight line, the reaction was classified as a pseudo first order reaction and the rate was determined by 2.303m (m = slope of line). If such a plot were not a straight line then i vs t was plotted. If this c were a straight line then a second order dependence was placed on the concentration of anhydrovitamin A. After the order and rate constants had been determined for anhydro vitamin A the order for the other reactants could be determined by a plot of log rate constant vs log of concen tration of fatty acid. The slope of this line determined the order with respect to the fatty acid. The reaction was allowed to proceed for ll+O hours at the specified tempera tures . Since anhydrovitamin A was being converted to other substances as well as to vitamin A as evidenced by the precipitation of the polymerization product, a plot of the log of concentration of anhydrovitamin A versus time would not show the true rate of vitamin A substance formation. Since the interest was in the rate of conversion of anhydro vitamin A into a vitamin A substance, the following procedure was used to obtain this data. A calculation for the extinction at 35lmu was made based on the assumption that all the anhydrovitamin A was converted into a vitamin A substance. This value was called A infinity ("A c>o ") and was calculated by multiplying the ratio of the values of vitamin A and anhydrovitamin A by the extinction of anhydrovitamin A at 35lmu as follows: 1 0 1 E1^ , vitamin A 1Cm (35lmu)------= _9C£ = 0.361 (1) Efcm (351ml) “ bydrovitamln A 2500 0.36lx E^5l (anhydrovitamin A) = A < W (2) Another value "A^" which was the extinction at 35lmu of vitamin A substance formed at any time t was also intro duced. Consequently it was believed that a plot of log (A C«0 -At ) versus time would give a good indication of the rate of anhydrovitamin A being converted into vitamin A. This procedure was thought to be desirable since it partially corrected the effects of anhydrovitamin A lost through other reactions. The rate of formation of vitamin A substance was reduced slightly; however, since only relative rates were desired, this procedure with cognizance of its limitations, was considered satisfactory. The plot of log (AOO- Aj.) versus t gave a straight line indicating a first order dependence on anhydrovitamin A. In order to determine the effects of the solvent alone on anhydrovitamin A, a control was prepared using anhydrovitamin A in cyclohexane without fatty acids. This was treated in the same manner as the other samples. The rate of change was negligible in the controls. It could be concluded that no vitamin A substance is formed in a system containing anhydrovitamin A and cyclohexane. The rates at 25>° are not recorded because they appear to be too slow to show any significant rate of change. 1 0 2 The pseudo first order rate constants are presented in Table 20. TABLE 20 RATES OP FORMATION OP VITAMIN A SUBSTANCE OP ACETIC ACID AND CAPROIC ACID - ANHYDROVITAMIN A IN CYCLOHEXANE k x (hr"1 x 10^-) Molarity 38° 50° 2M acetic acid 1.25 2.60 1+M acetic acid 2.08 l+.5o 6M acetic acid 3.90 7.50 2M caproic acid 1.11 3.33 caproic acid 2.37 5.85 6M caproic acid ij-.OO 12.5 The graphs of the rates at 50° are given in Figures 7 and 8 since they are considered to be representative of the group. The rate constants demonstrated that caproic acid had a faster rate of vitamin substance formation than acetic acid. When the logarithm of the k values in Table 20 were plotted versus log of concentration, the following slopes were obtained: H L 1 E S a t ADPITIOH OF tM BXBSBntUBM A TO CiJRPIC ACIP M g s OF ADOITICB OF u m m n o n v M aA to 1CP1C ACID « omiap*" »* *°* m ciiaoHm c at so* 0.1M M100 OIDP T IM ( TIM ( BOMS ) FIGURE a nsoag 7 1. Acetic acid at 50° = 1.05 2. Acetic acid at 38° = 1.09 3. Caproic acid at 50° = 1.1J? i|. Caproic acid at 38° = 1.19 This indicates essentially a first order dependence on the concentration of fatty acids in this system. It was desired to examine the effects of decanoic and palmitic acids on anhydrovitamin A in cyclohexane. Since these acids were not appreciably soluble in cyclo hexane, considerable difficulty was encountered. When the samples at 38° and 50° were drawn into a pipette for measurement, the fatty acids crystallized, consequently only qualitative tests were conducted. All fatty acids formed addition products with anhydrovitamin A as demonstrated by a paperchromatographic method described on page 105. Two chromatographic systems were tried in order to determine the better one. The paper was impregnated with 6 percent olive oil or 6 percent liquid petrolatum in petroleum ether (30° - 60°), with the same eluant in both cases; 50 percent methanol-ethanol. Certain observations were made with the first system: 1. Rf = 0.514-5 vitamin A acetate control 2. Rf = 0.076 vitamin A palmitate control 3. Rf = O.36 I anhydrovitamin A Lj.. The 2, L}., and 6M samples containing acetic acid and caproic acid contained vitamin A esters. 5. The zones of separation were not sharp. io 5 The problem of poor separation was solved by using the second system and the preliminary facts worth noting are: 1. Rf = 0.6Z|3 vitamin A acetate control 2. Rf = 0.060 vitamin A palmitate control 3. Rf = 0.320 anhydrovitamin A orange in U.V. light i^.. Vitamin A esters are yellow in U.V. light 5. Rf = 0.506 vitamin A caproate 6. Rf = O.I83 vitamin A decanoate 7. The 2, ij., and 6M samples contained vitamin A esters All the samples were extracted with 1 N NaOH solution to remove the fatty acids. The cyclohexane solutions were dried with anhydrous sodium sulfate and the solvent was removed in vacuo. The residue was dissolved in 5 ml* of petroleum ether and chromatographed on Type III Woelm alumina, in the usual manner, to isolate the ester. The solution of the ester was evaporated in vacuo to a concen trate and one drop was applied to l-§-,! Whatman Number 1 filter paper strips impregnated with a 6 percent solution of liquid petrolatum in petroleum ether. The strips were placed in a tank saturated with ethanol, methanol and nitrogen, and were allowed to develop for a period of J-Q hours, using equal parts of ethanol and methanol as the eluant. The identification of the esters was based on Rf values and U.V. absorption spectra. The typical vitamin A spectrum had a maximum at 326mu, which was considerably different from anhydrovitamin A, which had maxima at 351, 106 371, and 391mu. The results are summarized as follows: Sample: (l) Palmitic Acid-Anhydrovitamin A in Cyclohexane: (1) Rf = 0.309 Anhydrovitamin A (2) Rf = 0.060 Vitamin A Palmitate (2) Decanoic Acid-Anhydrovitamin A in Cyclohexane: (1) Rf = 0.312 Anhydrovitamin A (2) Rf = O.183 Vitamin A Decanoate (3) Caproic Acid Anhydrovitamin A in Cyclohexane: (1) Rf = 0.335 Anhydrovitamin A (2) Rf = 0.506 Vitamin A Caproate (I].) Acetic Acid-Anhydrovitamin A in Cyclohexane: (1) Rf = 0.320 Anhydrovitamin A (2) Rf = 0 .63 )+ Vitamin A Acetate These results showed that the anhydrovitamin A, which originally contained no esters, had been partially converted into vitamin A esters. It was desirable to determine the effects of the fatty acids on anhydrovitamin A in systems similar to those studied originally, namely, "99%", "95%", and "85%" isopropyl alcohol. The effects of water, fatty acid and isopropyl alcohol on anhydrovitamin A were observed in this series. The "99%" isopropyl alcohol solvent system was first studied. It consisted of the following mixtures: 1. Control - Anhydrovitamin A in "99%" isopropyl alcohol. 107 2. N/3 0 acetic acid-anhydrovitamin A In "99%” 1s opropyl alc ohol. 3. N/30 Caproic acid-anhydrovitamin A in "99%" isopropyl alcohol Ij.. N/3 0 decanoic acid-anhydrovitamin A in "99%" isopropyl alcohol 5. N/3 0 palmitic acid-anhydrovitamin A in "99%” isopropyl alcohol Since oxidation of the vitamin A produced would lower the rates of formation it was necessary to add 0 .1 percent of BHT as an antioxidant. The anhydrovitamin A was made by the method of Shantz (lip.) and was added to the previously pre pared samples of fatty acid in isopropyl alcohol which had been flushed with dry nitrogen for one hour. The samples were placed in 60 ml glass stoppered bottles and assayed by determination of extinction at 326 and 35lmu. The air above the samples was replaced by dry nitrogen, and the samples were placed in constant temperature apparatus at 37° and 50°. Each time a sample was removed for assay the air was dis placed by a stream of dry nitrogen to prevent oxidation. A logarithmic plot of (A© 0 - At) vs time was found to be a straight line, and first order with respect to anhydrovitamin A. This is logical since all of the in gredients were in a large excess as compared to anhydro vitamin A. The plots showed an initial rate, followed by a secondary rate. 108 Table 21 contains both rate constants of the system studied for a period of 280 hours* TABLE 21 RATES FOR TRANSFORMATION OF ANHYDROVITAMIN A INTO A VITAMIN A SUBSTANCE IN "99^" IS0PR0PYL ALCOHOL k x (hr”-*- x 10^-) "99%" Isopropyl N/30 N/30 N/30 N/30 Alcohol Acetic Caproic Decanoic Palmitic Control acid acid acid acid 5o° - 3.00 25.0 ki 3.75 ^•50 5.25 k2 1.11 1.21 1.314- 1.5 2.25 3?° kl 0.806 2.21 3.12 2.5 15.0 k2 0.52^ 0.835 0.925 1.13 2.00 In general the rate constants for the conversion of anhydrovitamin A into a vitamin A substance appeared to vary directly with the chain length of the fatty acid. It was also apparent that the rate of conversion into a vitamin A substance was always greater in the presence of the fatty acids than in the control. The control graph is submitted as representative of the entire group found in Figure 9. Since the previous study of vitamin A acetate in "95%" isopropyl alcohol showed faster rates of decomposition 109 of vitamin A than the "99%" isopropyl alcohol series, the effects of "95%" isopropyl alcohol on anhydrovitamin A in the presence of fatty acids were examined. The same method was used to prepare the samples as in the case of the "99%" isopropyl alcohol-anhydrovitamin A series. Precautions against oxidation were also taken. A plot of log (Ago - A.).) versus time was found to be linear, consequently the rates were calculated from the slopes of the lines. The control graph is presented as representative of the entire group (Figure 1 0 ). The equilibrium point appeared to be reached at 280 hours. The addition of five percent water to this system caused a significant increase in the rate constants which enabled the rates at 25° to be measurable. These rate constants are given in Table 22. Two distinct rates were not present, as observed with the "99%" isopropyl alcohol samples. This difference appears to be related to the increased amount of water present. Since the water produced an increase in the rate of vitamin A substance formation, a system containing an additional 10 percent of water ("85%" isopropyl alcohol solution) was examined. Exact water content was II4..36 percent by Karl Fisher method (97). The systems were prepared the same as the two previous ones. MnnWDVlTAMt A In "99 % • ISOPnnnrt. M M ieL COTROL OtSOO 02X00 T i m ( B M W ) now 9 ANHYDRO VITAMIN A IM " 95< * ISOPROPYL ALCOHOL CONTROL o H H i I X s» TBT TIMS ( HOURS ) FIOURK 10 Ill TABLE 22 RATES FOR TRANSFORMATION OF ANHYDROVITAMIN A INTO A VITAMEN A SUBSTANCE IN "95%" ISOPROPYL ALCOHOL k x (hr-1 x 10"^) "95%" Isopropyl N/30 N/30 N/30 N/30 Alcohol Acetic Caproic Decanoic Palmitic Control acid acid acid acid 5o° - k 2.09 3-5 2.86 3 .1 3.33 37° - k O.83I4. 1 .67 2 .11|. 1 .37 1 .51+ 25° - k 0 . 1+2 0.£0 0.83 0 . 1+2 0 . 1+2 An increased rate of formation of vitamin A active substances was noted over the previous systems. At 50° the samples reached equilibrium at lipO hours . The plot of log (Aoo - A^) versus t appeared to be a straight line and followed the logarithmic rate law. For the first time the rates at 50° of the control, decanoic and palmitic acid samples were almost equal and the fatty acids failed to produce a greater rate of vitamin A suostance formation than the control. There was evidence to show larger amounts of oxidation in these samples and this may explain the de creased rate. The rates are given in Table 23. 1 1 2 TABLE 23 RATES FOR TRANSFORMATION OF ANHYDROVITAMIN A INTO A VITAMIN A SUBSTANCE IN "85$" ISOPROPYL ALCOHOL k x (hr-1 x 10^) "85$" Isopropyl N/3 0 N/30 N/30 N/30 Alcohol Acetic Caproic Decanoic Palmitic Control acid acid acid acid 50° - k 1+.55 8.00 5.72 1+.91+ 1+45 37° - k 2.22 6.11-5 1+45 1+43 il. 00 25° - k 0.863 1.11 0.871+ 1.6 1.56 The control graph is submitted as representative of the entire group, Figure 11. Paper chromatography was used to examine the reaction products. Whatman filter paper, No. 1 (l'J-") impregnated with a 6 percent solution of liquid petrolatum in petroleum ether 30-60°, with ethanol USP as the eluant, was used according to the procedure described under the chromatography of vitamin A acetate in "95%” isopropyl alcohol. A set of controls was made to compare the R^ values of the known substances to those found on the strips. LOO ( A*>- At x 10* aisoo- 0.1000 - ANHYDROVITAMIN ANHYDROVITAMIN A IS OR ) ( HOORS TIHS IN * 85# " ISOgROPYL ALCOHOL CONTROL ALCOHOL ISOgROPYL " 85# * IN FIGURE 11 FIGURE Mb 113 Ilk Control Rf Values 1 . Vitamin A palmitate 0.070 2 . Vitamin A decanoate 0.200 3- Anhydrovitamin A 0.370 Vitamin A caproate 0.526 5. Vitamin A acetate 0.630 6 . Vitamin A alcohol 0.930 The Rf values found In the systems containing "99%" isopropyl alcohol and anhydrovitamin A are listed in Table 2k* TABLE 21+ Rf VALUES OP SUBSTANCES POUND IN "99$" ISOPROPYL ALCOHOL-ANHYDROVITAMIN A SYSTEMS, "99%" Isopropyl N/3 0 N/3 0 N/30 N/30 Alcohol control Acetic Caproic Decanoic Palmitic acid acid acid acid O.36 O 0.375 0.382 0 .200* 0.067* --- 0 . 6 1 5 ' “' 0.550* 0.390 0 .381). 0.739 0.712 0.735 0.719 0.696 0.930* 0 .921+* 0 .91+1'“' 0.920* 0.935* *These substances possess a typical vitamin A U.V. spectrum (maxima at 325>-328mu). The chromatographed "99%" isopropyl alcohol systems all showed (l) zones with an Rf value of 0.360-0.390 characteristic of unreacted anhydrovitamin A, (2) zones 115 in the vicinity of 0.920-0.9l]l characteristic of vitamin A alcohol, (3) zones at 0.696-0.739 with a spectrum of oxidized vitamin A. In the fatty acid solutions small zones of vitamin A active material corresponding to the Rf value of the vitamin A ester formed from the fatty acid of the media were found. The samples in "95$” and "85%" isopropyl alcohol were very similar in content and could be described simul taneously by the "85%" isopropyl alcohol samples. The results are given in Table 25* TABLE 25 Rf VALUES OP SUBSTANCES POUND IN "85%" IS0PR0PYL ALCOHOL-ANHYDROVITAMIN A SYSTEMS "85%" Isopropyl N/30 N/30 N/30 N/30 Alcohol control Acetic Caproic Decanoic Palmitic acid acid acid acid 0.39k 0.390 0.381 0.221]* 0.072* --- 0.610* 0.552* 0.1].00 0.395 0.781 0.761]. 0.773 0.757 0.790 0.925* 0.929* 0.916 0.900* 0.930* "^Exhibits the U.V. spectrum of vitamin A type substance. In these latter two cases, the pattern is the same as in the "99%" isopropyl alcohol samples. A mixture of 1 1 6 anhydrovitamin A, vitamin A alcohol, vitamin A esters formed from the fatty acid of the media and oxidation products was found. The quantity of vitamin A alcohol in these hydro alcoholic systems appeared to be greater than in the "99%" isopropyl alcohol systems and varied directly with the percent water in the media. PREPARATION OF SOME NEW ESTERS OF VITAMIN A Introduction There is considerable information regarding the action of lower alkyl alcohols and strong acids on vitamin A alcohol and its derivatives. The main reaction appears to be an eliminative degradation with the formation of anhydro- vitamin A. Shantz (lip.) found that vitamin A esters, when refluxed in ethanol, formed a mixture of anhydrovitamin A, fatty acid, vitamin A ethyl ether and other products. It was also stated that anhydrovitamin A could be made faster if the system contained an acid catalyst such as N/30 HC1. This catalyzed reaction was found to be much faster with vitamin A alcohol than with the esters. Anhydrovitamin A can be synthesized from vitamin A alcohol by treating it with N/3 0 HCl in anhydrous ethanol (lip.). Prolonged contact of anhydrovitamin A with ethanolic HCl causes the formation of a substance called isoanhydro- vitamin A (lip.) by the addition of a molecule of ethanol 117 across one of the terminal double bonds. Meunier (H 4.O) described the "possible” mechanism of (H+) catalysis of anhydrovitamin A formation from vitamin A alcohol. /7 ^ +HC)i No information has been found in the literature regarding the mechanism of elimination of vitamin A esters. It is an accepted fact that (H+) catalyzed elimination in vitamin A esters is slower than in vitamin A alcohol. An examination of the theoretical mechanism of elimination in vitamin A alcohol suggests an analogous situation: •f^CooH-tycoon — This appears to be reasonable in light of Shantz’s (lip.) observation that refluxing the ester with ethanol gives anhydrovitamin A plus fatty acids. It also appears logical that the greater stability of the ester against acid catalysis might be connected with the electronegative effect of the carbonyl group adjacent to the alkyl oxygen causing a reduction of the electron density around the latter group. The attracting forces of the proton to that alkyl oxygen and 118 the Initial step of the elimination reaction are consequently reduced. The steric blockade and attraction of the carbonyl oxygen may also have a bearing on the greater stability of the esters as compared to vitamin A alcohol. In the present study it was thought desirable to synthesize a series of esters having electronegative groups in the alpha and beta positions of the fatty acid group. This was based on the premise that a reduction of the electron density around the alkyl oxygen should render it less sus ceptible to proton attack, and giving esters that are more stable against eliminative degradation. Two series of compounds were synthesized: 1. Compounds having chlorine in the o i position of the fatty acid, such as vitamin A trichloroacetate, chloro- acetate, and chloropropionate. 2. Compounds having unsaturation in the 2 position of the fatty acid, such as vitamin A acrylate, crotonate and sorbate, the latter also having unsaturation in the I}, position. Table 26 shows that the acids used in esterification have larger Ka's than the saturated unsubstituted acids from which they are derived. The esters were prepared and their stability was studied in the following media: 1. HCl in anhydrous ethanol 2. HCl in ethanol U.S.P. 3. UaOH in ethanol U.S.P. 119 Isopropyl alcohol and hydro-isopropyl alcohol solutions. 5. The systems mentioned in 1^. containing a fatty acid. TABLE 26 Ka OP ACIDS RELATED TO THIS STUDY AS GIVEN BY INGOLD (L|i|) Acid Ka x 105 Acetic acid 1.75 Chloroacetic acid" 155.0 Dichloroacetic acid 5,100 Trichloroacetic acid-* 120,000 Propionic acid 1.3^ Ghloropropionic acid* lltf '0 Acrylic acid"'1- 5.56 Butyric acid 1 .14-8 Crotonic acid* 2.03 Caproic acid 1.32 Sorbic acid 1.73 ''Esters containing these acids were synthesized. 1 2 0 SYNTHESIS Preliminary Data The first syntheses to be attempted were the chloro derivatives according to the metnod of Baxter and Robeson (155)* This method essentially entails treating vitamin A alcohol with the acid chloride in the presence of pyridine as a proton scavenger to remove the HCl formed as the precipitated pyridine hydrochloride. The reaction is represented as follows: This reaction mixture was allowed to stand in the dark for 2 hours and was poured into 0.5 N HgSO^ to remove any unreacted pyridine. The solution of the ester was sub sequently washed with 10 percent Na2C0^ solution to remove any unreacted acid chloride then with distilled water and dried with anhydrous Na2S0^. The solvent was removed in vacuo and methanol was used as the crystallizing solvent. When this procedure was used the trichloroacetate could not be made and low yields were obtained with the other two esters. It appeared that contact with water had a destructive effect on these compounds. These esters could \ not be crystallized from methanol because that solvent catalyzes elimination. In order to obtain more information about the products of the above reaction they were subjected to a variety of tests: 121 1. Product from attempted vitamin A trichloroacetate synthesis: (a) U.V. spectrum - maxima at 351 > 371> 391mu (anhydrovitamin A). (b) I.R. spectrum - hydrocarbon; no carbonyl group. (c) Chromatography on alumina confirmed presence of anhydrovitamin A as major reaction product. 2. Product from vitamin A chloroacetate synthesis: (a) TJ.V. spectrum - maxima at 326, 35l> 371> 391mu (vitamin A and anhydrovitamin A). (b) I.R. spectrum - carbonyl group. (c) Chromatography on alumina - separation of a compound with properties of vitamin A ester and U.V. maximum at 326mu. 3. Product from vitamin A o6 chloropropionate synthesis: (a) U.V. spectrum - maxima at 326, 351» 371» 391mu (vitamin A and anhydrovitamin A). (b) I.R. spectrum - carbonyl group. (c) Chromatography on alumina - compound with adsorption properties of vitamin A ester, and U.V. maximum at 326mu. When the alumina chromatographed fractions in (2) and (3 ) were subjected to paper chromatography a mixture of vitamin A alcohol, anhydrovitamin A and vitamin A ester separated on the paper. The alcohol was not originally present since the I.R. spectrum showed no alcohol group. 122 Consequently it was assumed that these esters were very sensitive to hydrolysis and elimination, which probably caused the low yield in the presence of water. It became obvious that these esters could be made; however traces of water could seriously affect them, the effect being most pronounced with the vitamin A trichloroacetate. A method was devised to make these esters and purify them in a system where the water would not be available. Vitamin A alcohol, fatty acids, and acid chloride are more strongly adsorbed on alumina than the vitamin A ester and anhydrovitamin A is least strongly adsorbed. Consequently if the whole reaction mixture was poured through an alumina column, the ester could be separated. Pyridine traveled at the same rate as the ester, consequently the mixture was subjected to vacuum distillation at a low temperature to remove the excess pyridine. General Method of Preparation of Esters This method was used for all of the esters; modifi cation will be discussed under individual compounds. Pour and one-half grams (0.016M) vitamin A alcohol was dissolved in 25 ml ethylene dichloride containing 5 ml of dry pyridine and was cooled to 10°. In a separate flask, 0.018M of the acid chloride was dissolved in 25 ml of ethylene dichloride. While the vitamin A solution was being stirred with a magnetic stirrer the acid chloride solution was slowly added and kept in a dark place for 2 hours. 123 The solvent was then removed in vacuo and the residue dissolved in petroleum ether (30°-60°). Woelm alumina was deactivated by the addition of 8 percent water; a slurry was made of the deactivated alumina in petroleum ether (30°-60°) and poured into the column. Fifty grams of alumina were used for each gram of ester. The reaction mixture was poured through the column followed by petroleum ether (30°-60°). The first substance eluted was anhydrovitamin A and it was followed by the vitamin A ester. Anhydrovitamin A is easily distinguished from the ester because it has an orange fluorescence in U.V. light, whereas the ester has a yellow green color. The fraction containing the ester was evaporated under reduced pressure, using a nitrogen bleeder, then the residue was dissolved in petroleum ether (30°-60 °) and rechromatographed on the same type of alumina. The petroleum ether was first evaporated and then the pyridine was removed by distillation under 0 .5mm pressure for a period of four hours and yellow viscous oils were produced. Attempts to crystallize the ester from a variety of polar, nonpolar or mixtures of the two types of solvents were not successful. In order to obtain the correct polar, nonpolar solvent combination, the following solvents were tried: petroleum ether (BP 30°-60°), benzene, ethyl ether, ethyl acetate, acetone, chloroform, ethyl ether-petroleum ether (3O0-60°), acetone-ethanol, petroleum ether (30°-60°) - acetone and petroleum ether (30°-60°) - benzene. These 124 solutions were placed at -15° for crystallization; dry ice- acetone baths at -60° were also used. Solutions containing benzene were not cooled below 6°. After four months no crystals developed. Materials Vitamin A Alcohol Roche MW 286 Ghloroacetyl Chloride Eastman (redistilled) MW 112.95 Trichloroacetyl Chloride Eastman (redistilled) MW 181.85 0 ( - Chloropropionyl Chloride Eastman (redistilled) MW 126.98 Pyridine dried over CaCl2 (redistilled) Ethylene Dichloride Eastman Woelm Neutral Activated Alumina Petroleum Ether B.P. 30°-60° Mallinckrodt (AR) Vitamin A Chloropropionate (M.W. = 376.5 Gm/M): Following the general method previously described 8 gm (0.0284 M) of vitamin A alcohol and ij.• 061). gm (0.0313 M) of the o C chloropropionyl chloride gave 10.0 gm ester; 93*85 percent yield. Vitamin A monochloroacetate (M.W. 362.5 Gm/M): Following the general method previously described 8 gm (0.0284 M) of vitamin A alcohol ana 3*57 gm (0.0313 M ) of the chloroacetyl chloride gave 8.35 gm ester; 8l.O percent yield. Vitamin A trichloroacetate (M.W. 4-31*0 &n/M): Following the general method described 6 gm vitamin A 125 alcohol (0 .0213M) and L\.,3t>k- gm (0 .02l|0M) of trichloroacetic acid chloride were used. An almost quantitative yield of 5.57 gm anhydrovitamin A equivalent to about 95 percent of the vitamin A was obtained but no ester. It appeared that vitamin A trichloroacetate was so unstable that it could not be made by this method. Vitamin A Sorbate (M.W. 38I.O G-/M) : Sorbic acid chloride was prepared from sorbic acid as follows: 18.0 gm of sorbic acid (0.1765 M), M.W. 112, was placed in a triple necked 250 ml flask equipped with a mag netic stirrer, 58 ml of benzene were added to make a slurry and a reflux condenser was attached. One gram of C^Cl^ was added to reduce polymerization. Thirty-one and one-half grams (0.26L|.7M) of thionyl chloride were added dropwise and mixture was stirred for two hours, then refluxed over gentle heat for 30 minutes. The thionyl chloride, benzene and HCl were removed by distillation under the reduced pressure of a water pump and the residue was distilled at 110° bath temperature; the acid chloride was collected at 79°-80° 13mm. Using the general method, 8 gm (0.0281|M) of vitamin A alcohol and I4..176 gm (0.032M) of sorbic acid chloride yielded 7.5 gm of ester (80.25$). A purple color was formed in the reaction media which did not affect the esterification. The general method of preparation could not be used for the preparation of vitamin A acrylate and crotonate. The acid chlorides immediately reacted with pyridine to give 126 a material which precipitated from the ethylene dichloride solution. They were thought to be the quaternary salts of pyridine, since they were easily water soluble. This reaction was undesirable for two reasons: 1 . It removed the pyridine which was necessary for protection against acid. 2. It removed the acid chloride which was needed to react with the vitamin A alcohol. Other amines were substituted for the pyridine. Trimethylamine was first used, and was round to be unsatis factory. Since pyridine has a = 2.3 x 10“9, bases with lower Kb >s were tried. Quinoline = 1 x 10“^ was satis factory for vitamin A crotonate and dimethylaniline, = 2.1+2 x 10"-*-® was required for vitamin A acrylate. Vitamin A Crotonate (M.W. 35i]-»0 G/M): Using the general method with the substitution of quinoline for pyridine, 9.0 gm (0.032 M) of vitamin A alcohol and 3*762 gm (O.O36 M) of the crotonic acid chloride gave 6.0 gm of ester; 53 percent yield. Since quinoline boils at 216 ° it was removed by distillation at 60 °-70° for 6 hours under 0.5 mm pressure. This treatment may have increased the decomposition of the ester. Vitamin A Acrylate (M.W. 3^0.0 G/M): Using the general method with exception that di me thylaniline replaced the pyridine, 10 gm (0.0356 M) vitamin A alcohol and 3*619 gm (0.0399 M) acrylic acid 127 chloride yielded 5*5 g™ of ester; !l5«5> percent yield. Since dimethylaniline boils at 197° it was necessary to subject the residue to distillation at 60 °_70° at 0.5 ram for a period of 5-6 hours to remove the dimethylaniniline causing low yields. Infra Red Spectra These compounds were subjected to I.R. and U.V. spectrum analysis. The I.R. graphs found in Figures 12 and 13 were obtained on a Baird Infrared Spectrophometer. The I.R. spectra showed 1 . a definite presence of ester groups 2 . the absence of hydroxyl groups 3 . the absence of quinoline, pyridine and dimethyl aniline. The carbonyl peaks for these compounds were different from each other, consequently they are tabulated in the following table as a possible means of identifi cation. PERCENT TRANSMRTANCE 8 PERCENT * TRANSMITTANCE S S 8g ©8888 PERCENT TRANSMITTANCE 1 ------1------i------5------r g ------1------1------1------1------r AE 06H M L046TH WAVE WAVE NUMKRS WAVE -tt WAVE LB4GTH WAVE LENGTH IN MICRONS WAVE NUMIERS M CM-' WAVE NUMBERS IN CM-1 WAVE LENGTH M ktC R O N S WAVE LENGTH IN kNCRONS WAVE NUMBERS IN CM-' WAVE LENGTH F I G - U . R E R 130 TABLE 27 INFRARED CARBONYL PEAKS OF NEW ESTERS IN {JLL) Ester Position of Peak Vitamin A ^chloropropionate 5.71 Vitamin A chloroacetate 5.6l|. Vitamin A sorbate 5.80 Vitamin A crotonate 5.76 Vitamin A acrylate 5.7i| Vitamin A palmitate'"' 5.73 ':s-This compound was included for comparison. U.V. Spectra In order to determine the accurate U.V. extinction coefficients, each of the new esters was rechromatographed prior to extinction studies. The solvent in these deter minations was petroleum ether (3OO-6 O0 ) Mallinckrodt (A.R. grade), a Beckman D.U. spectrophotometer and silica cells were used. The coefficient was determined by the following equation and the results are given in Table 28. "L% ^lcm(max) = ,(°b3 ). C = percent solution C L L = length of cell in cm 131 TABLE 28 EXTINCTION COEFFICIENTS OF THE NEW ESTERS E s t e r E 1 ^ m a x ( m u ) 1cm Vitamin A chloroacetate l O i j . 6 325 Vitamin A p^chloropropionate 1300 328 Vitamin acrylate 1387 327 Vitamin crotonate 1318 327 Vitamin A sorbate 1318 327 The low extinction of -vitamin A chloroacetate immediately after purification indicates its lack of stability. During its purification substantial amounts of anhydrovitamin A were removed. It is quite likely that more had formed between the time of purification and spectral analysis. The graph of the complete U.V. spectrum illus trates this point. In order to determine the purity, and to observe if these compounds possessed the typical U.V. spectrum of vitamin A, the U.V. spectral analyses were made in petroleum ether (30°-60°) (Mallinckrodt, A.R.) on a Cary Recording Spectrophotometer. All the new esters had a typical vitamin A spectrum, maximum (325-328mu) except vitamin A chloroacetate; although purified immediately before analysis it showed impurity maxima at 360, 3J4I and 310mu. The vitamin A sorbate had 132 2 peaks, one due the chromophore of the vitamin A molecule (326mu) and the second due to the sorbate portion (255>mu). The U.V. spectrographs are given in Figures llp-l8. Determination of Rf Values Since Rf values have been used as a means of identify ing compounds it was desired to determine the Rf values of these esters along with already existing forms of vitamin A compounds(Table 29). The method was described under paper chromatographic separation of vitamin A acetate in U9B%" isopropyl alcohol samples. The lipophile-eluant combination was 6 percent liquid petrolatum-(ethanol U.S.P.). The new esters with the exception of vitamin A chloroacetate showed only one spot on the paper, indicating pure substances. The chloroacetate ester appeared to be a mixture of the ester and anhydrovitamin A, which indicates the sensitivity of this ester to ethanol. 13 3 1200 ULTU V10LST.ABa0RPTIQM.ArftfiSRUH_£r VIT^/vIX A CKlDROASSTAtL ii» ?&TROLrj;: jcthsr ( ?o°- 60°) poo UJ ?00 Wavelength (091) Figure 1* PLTRA YIOU? A3S0RP7ICK SFE??3UK OF VITAXIW A«CC:iLCRO?RO?ICrAT£ IK FSTROlgJM OTHER ( ^0°- *0° ) 700 35C ULT3/. VICLKT ABaCRfTIOa S.^CTRUM Q? VITAKIN A ACRYLATa IK PiTROLEUX STHER ( 3C°- 60°) 1400 1050 320 380 V&vslanpth (op) Mgur«_16 1600. ULTRA V I O U T A1S0RPTI0N SPESTR7K OF-VITAMIN A CROTCNATS IN PSTROLSUM ^TH£.4 ( 3Cf- 60°) 1200. 800 400 I35- 1 4 0 0 f1050 f 3 5 0 I VITAMIN A SO RBA TE iR ~RnRoUuw EtHeR ('3o°-faQ‘') OF OF ULTRA VIOL.&T ABSORPTION SPECTRUM Ftgiura Id 136 TABLE 29 Rf VALUES OP VITAMIN A ESTERS AND RELATED COMPOUNDS Compound Rf value Neovitamin A alcohol 0.960 Vitamin A alcohol 0.920 Vitamin A methyl ether 0.725 Vitamin A acetate 0.651 Vitamin A acrylate 0.620 Vitamin A sorbate 0.570 Vitamin A crotonate 0.550 Vitamin A chloroacetate 0.525 Vitamin A chloropropionate o.5i5 Vitamin A palmitate 0.100 Stability of Esters After the identity of these esters had been established, it was decided to study their stability in the following reagents: 1. 0.01N NaOH in ethanol USP 2. 0.0113N HCl in anhydrous alcohol 3. 0.0113N HCl in ethanol USP [).. "99%" and "95%" isopropyl alcohol 5. "99%” and "95%” isopropyl alcohol with N/30 decanoic acid. 137 Stability Against Base Two meq. of ester were accurately measured and dissolved in 50 ml of 0.01N NaOH in ethanol USP. The rates of saponification were studied at 10° * 1°. The rate of disappearance of base was measured by titrating the solution with 0.01N HCl using thymol blue indicator. Since the speed of depletion of base is an indi cation of the stability against that reagent, the time required to consume 1.0 ml base are listed in Table 30. TABLE 30 TIME REQUIRED TO CONSUME 1 ml OP 0.01N NaOH Ester Time (minutes) Vitamin A chloroacetate 0.85 Vitamin A chloropropionate 2.10 Vitamin A acetate*' 9.5 Vitamin A palmitate*' 118.0 Vitamin A acrylate 170.0 Vitamin A sorbate 268.0 ’’''“These compounds were included for purposes of comparison. Table 30 indicates that the new chloro esters are very sensitive to base hydrolysis, whereas the unsaturated 138 acid esters are more resistant than either the acetate or palmitate esters. The saponification equivalents of these esters were determined by the method of Shriner and Fuson (95)* After refluxing the esters with base the solutions turned dark brown and potentioraetric titrations were used. The results are listed in Table 31. TABLE 31 SAPONIFICATION EQUIVALENTS OP ESTERS Molecular Saponification Percent Ester weight equivalent variation G/M g/m from theoretical Vitamin A chloroacetate 362.5 329.5 -9.13 Vitamin A oC c hior oprop i onat e 376.5. 385.)+ +2.61 Vitamin A acrylate 311-0.0 3)1-7.33 +2.15 Vitamin A crotonate 35)1-.o 362.01 +2.21 Vitamin A sorbate 381.0 385.82 +1.27 The unusually low saponification equivalent and negative percent variation from theoretical of vitamin A chloroacetate indicated the presence of a lower molecular weight substance than the ester. This could be possible since only the U.V. spectrum of this compound indicated the presence of other substances beside vitamin A. 139 Stability of the Eaters In 0.0113 N HCl in Anhydrous Ethanol Anhydrous ethanolic HCl was prepared by bubbling dry HCl through anhydrous ethanol and was diluted to a concen tration of (0.0113N). It was found to contain O.II4.8 percent water by the Karl Fisher method (97)- A quantity of concen trate of the vitamin A substance was added to the acid solution so that it would have a U.V. extinction of approximately 0.700 at 326mu. The solution was transferred to silica cells and put into the spectrophotometer. The temperature of the water flowing through the hydrogen lamp housing of the spectrophotometer was set at 15° + 1° and the cells remained in the instrument throughout the entire series of measurements; extinction was measured at 326 and 391mu. Since anhydrovitamin A has an extinction at 326mu equal to 0.2 E^91J ^his amount was subtracted from E326mu to give a more valid measurement of the vitamin A content at any time t. A plot of log obs “ °»2 E39i obs^ vs ^ was a straight line and followed a logarithmic rate law. The ethanol, HCl and water were in large excess and did not affect the rate equation. The rates are given in Table 32. li+O TABLE 32 RATES OP ELIMINATION OP VITAMIN A ESTERS IN 0.0113 N HCl (ANHYDROUS ETHANOL*) Vitamin A substance k x (10^- min.■"-*-) Vitamin A chloroacetate Too fast to measure Vitamin A alcohol 150.0 Neovitamin A alcohol 65.0 Vitamin A methyl ether 23.0 Vitamin A sorbate 15.0 Vitamin A acetate 12.5 Vitamin A palmitate 9.62 Vitamin A crotonate 9.35 Vitamin A acrylate U-.77 Vitamin A Q^chloropropionate 2.32 '“Water content O.IJ4.8 percent. The rates of elimination of these esters in 0.0113 N HCl-ethanol USP were determined, using the same testing procedures and correction factors as above. The results are given in Table 33* TABLE 33 RATES OP ELIMINATION OP VITAMIN A ESTERS IN 0.0113 N HC1 (ETHANOL USP) Vitamin A substance k x (10^ min.”^-) Vitamin A chloroacetate Too fast to measure Vitamin AO^ chloropropionate 68.2 Vitamin A alcohol 66.0 Vitamin A methyl ether 11.5 Vitamin A acetate 11.0 Vitamin A sorbate 6.9 Vitamin A crotonate 6.0 Vitamin A palmitate 3.0 Vitamin A acrylate 2.0 It was observed that the chloropropionate ester was the most stable ester in anhydrous ethanolic HC1, however when water was added it became very unstable. This was probably due to the solvolytic action of water which was not present in the anhydrous solution. Stability of Esters in Isopropanol Plus Fatty Acids Since the acetate and palmitate had been tested for stability in isopropyl alcohol solution of fatty acids, the new esters were studied under the same conditions. The limited quantity of ester only permitted investigation with 1 i|2 one fatty acid. Since previous results showed that there are no considerable differences in the action of the different fatty acids the use of only one fatty acid was satisfactory. The samples consisted of vitamin A ester in ”99$" isopropyl alcohol and ”95$" isopropyl alcohol with and without N/30 decanoic acid. The solutions were prepared and handled in the same manner as the previous series containing esters and fatty acids. The assay method was that of Gama (128). Vitamin A acrylate. Vitamin A acrylate was dissolved in isopropyl alcohol and divided into four equal parts. Adequate portions were added to ”99$" isopropyl alcohol, "95$" isopropyl alcohol, N/30 decanoic acid in "99$" isopropyl alcohol and N/30 decanoic acid in "95$" isopropyl alcohol respectively and each was divided into 3 parts and stored at 25°, 37°, and i|5°. The rates of decomposition of the esters are given in Table 3l+ and the graph of the rates is found in Figure 19. LOO CONCENTRATION 0T VITAMIN A LOO CONCENTRATION 0? V I T A M I N A to- - o a R s- Co § CxJ lljij. TABLE 3l|. RATES OP DECOMPOSITION OP VITAMIN A ACRYLATE IN ISOPROPYL ALCOHOL k x (103 hr.”1 ) 25° 37° k$° 25° 37° k$° "99$" Isopropyl Alcohol "95$" Isopropyl Alcohol k-L l+.lo 5.75 7.50 10.0 12.5 22.2 k2 1-25 1.97 3.00 1.88 2.88 I|..77 N/30 Decanoic Acid "99$" N/30 Decanoic Acid "95$" Isopropyl Alcohol Isopropyl Alcohol k-L 1.72 3.00 5.00 5.00 10.00 10.00 k2 1.18 1.88 1.88 1.67 2.72 l|..ll ■^1 represents the first ten hours of decomposition and k£ represents the rate from that point on. Vitamin A chloroacetate. The solutions were pre pared as directed under vitamin A acrylate. This ester showed the fastest rates of decomposition, consequently only the initial rates were measurable. The graph of the "99$" isopropyl alcohol control is found in Figure 20 and the rates are in Table 35. 1k5 TABLE 35 RATES OP DECOMPOSITION OP VITAMIN A GHLOROAGETATE IN ISOPROPYL ALCOHOL k x (102 hr."1 ) 25° 37° ij£° 25° 370 1+5° "99$" Isopropyl Alcohol "95$" Isopropyl Alcohol 2.20 5.00 8.5 2.00 Ip.32 k-32 N/30 Decanoic Acid "99$" N/30 Decanoic Acid "95$" Isopropyl Alcohol Isopropyl Alcohol 2.75 5 .0 5.9 i^.OO 7.5 8.3 Vitamin A sorbate. The samples were prepared as directed under vitamin A acrylate. The rates are given in Table 36 and the control graph Is given in Figure 21. £ (vi/wi) LOG CO"ChiNTRATICU OF 7ITAMTN A TIME f HOUH3 ) FIGURE 21 VITAMIN A oC CHLOROPROPIONATE In LOO CONCENTRATION OF VITAMIN A ( U/al.) A ( VITAMIN OF CONCENTRATION LOO e-- TIMS ( HOURS ) f i g u r e 82 TABLE 36 RATES OP DECOMPOSITION OP VITAMIN A SORBATE IN ISOPROPYL ALCOHOL k x (103 hr.”1 ) 25° 37° 25° 37° b$° "99$" Isopropyl Alcohol "95$" Isopropyl Alcohol (control) (control) kl 1 .6 6 2 .0 8 ip. 16 1.07 3.33 5 .0 0 k 2 0 .5 3 1 .3 6 l . ip7 2.0k 2.1p3 N/30 Decanoic Acid "99$" N/ 3 0 Decanoic Acid "95$ " Isopropyl Alcohol Isopropyl Alcohol kl 0.125 O.lpLO 1 .6 6 O.33 O 1.53 3 .2 2 k2 Vitamin A chloropropionate. The samples were prepared as directed under vitamin A acrylate. The rates of decomposition are given in Table 37* The graph of the ”99$" isopropyl alcohol control is in Figure 22. llj.8 TABLE 37 RATES OP VITAMIN A CHLOROPROPIONATE DECOMPOSITION IN ISOPROPYL ALCOHOL k x (103 hr."1 ) 25° 37° 1*5° 25° 37° U5° "99% " Isopropyl Alcohol "95%" Isopropyl Alcohol (control) (control) kl 10.00 33-3 56.1 17.0 63.5 8 3 .5 k2 2.5 2.75 kl 7-5 27.7 5 0 .0 12.0 5 0 .0 80.0 k2 2.25 1.0 These rates demonstrate the extreme sensitivity of vitamin A chloropropionate to solvolysis. The addition of 5 percent water practically doubles the rate of decomposition. Only the 25° samples showed 2 rates. The 37° and 1|5° samples appeared to reach equilibrium at 5 hours. Vitamin A crotonate. The samples were prepared as directed under vitamin A acrylate. The graph of the "99%>" isopropyl alcohol control is in Figure 23 and the rates of decomposition are given in Table 38* li+9 TABLE 38 RATES OP DECOMPOSITION OP VITAMIN A CROTONATE IN ISOPROPYL ALCOHOL k x (lo3 hr.-1) 2$° 370 k$° 25° 370 1+5° "99%" Isopropyl Alcohol "95$" Isopropyl Alcohol (control) (control) ^ l.ii.3 2.95 5.75 1 .8 8 3 .1 3 6 .6 6 k2 O.33I4. 1 .0 3 1 .3 7 /,X 0 .3 6 8 ° 1.67 3.71 N/30 Decanoic Acid "99%" N/30 Decanoic Acid "95%" Isopropyl Alcohol Isopropyl Alcohol kx 0.278 1.19 3-13 0 .14.03 I.L4.7 k2 0.200 1.15 ° p o JO o g'pr. 00 nao g° cSo 0$ ■ c 0 '0 0 % ^ 0 C? 3 • c Chromatography of .the Reaction, Mixtures” 0 " °, ’ ’ q 0 v- o o O In order to identify the compounds formed in these reaction mixtures, they were subjected to paper chromato graphy using Whatman filter paper No. 1, 1-g-", impregnated with 6 percent liquid petrolatum in petroleum ether (30°-60°). The papers were eluted with ethanol at USP for 7 hours at 30°. The reaction samples at 25° and 37° were examined. The identification of the spots was based on their Rf values and U.V. spectrum in ethanol USP. *(00 CONCENTRATION OF VITAMIN A ( O/ml.) a as ro £ O f H |M OS! 151 A series of controls had the following: Rf values: Vitamin A alcohol 0.910 Vitamin A acetate 0.651 Vitamin A acrylate 0.620 Vitamin A sorbate 0.570 Vitamin A crotonate 0.550 Vitamin A chloroacetate 0.525 Vitamin A o(chloropropionate 0.515 Anhydrovitamin A 0.350 Vitamin A decanoate 0.260 Vitamin A palmitate 0.100 The TJ.V. spectroscopic properties of the compounds in these systems were: Vitamin A and esters - maximum 325“328mu. Anhydrovitamin A - maxima 351 > 371 and 391mu. , Oxidation products - 310, 290, 275mu. The samples appeared to have a similar composition. • They all contained anhydrovitamin A, vitamin A alcohol, the ester originally introduced, and oxidation products. The N/30 decanoic acid solutions of the acrylate, crotonate and sorbate contained small quantities of vitamin A decanoate and larger amounts of vitamin A alcohol than the controls without fatty acids. 152 DISCUSSION Effect of Fatty Acids The original purpose of this study was to determine the effects of fatty acids on vitamin A esters. The interest in this subject was prompted by the report that vitamin A esters were unstable in vanishing cre-am bases containing fatty acids (2). Since vanishing cream bases contained a variety of ingredients, it was desirable to study this reaction in a single solvent system. Isopropyl alcohol was chosen as the solvent because it has only a slight de structive action on the vitamin, was water miscible and was a good solvent for vitamin A. Caproic, decanoic, palmitic, stearic and oleic acids have been shown to have a stabilizing "action on vitamin A palmitate in "99^” isopropyl alcohol when compared to a control not containing fatty acids. The addition of 5 per cent water to the "99%" isopropyL alcohol systems increased the rate of elimination of the esters, however the fatty acids still retained some of theip stabilizing action when compared to the control. The effect of water in these systems was in agreement with the results of Higuchi and Reinstein (56) who found that the rate of elimination of vitamin A acetate in hydroalcoholic solutions varied directly with the water content. The chromatographed isopropyl alcohol-ester-fatty acid reaction mixtures demonstrated that there were forms of vitamin A present which were not originally introduced. A 153 solution of vitamin A acetate in N/ 3 0 palmitic acid-"95/£" isopropyl alcohol contained a small quantity of vitamin A palmitate. The other fatty acid solutions also contained vitamin A esters formed from the fatty acid of the reaction media. Action of Fatty Acids The control solutions (without fatty acids) in variably contained more anhydrovitamin A than the fatty acid solutions. Thus the increased stability of the esters and smaller amounts of anhydrovitamin A in the presence of fatty acids could be caused fcy two reactions which are both possible: 1. Suppression of anhydrovitamin A formation by a common ion effect 2. Reaction of the fatty acid with anhydrovitamin A to reform vitamin A ester or vitamin A substance It has been theorized by Embree (121+) that the initial step of vitamin A alcohol degradation in acid is the formation of the carbonium ion. It is possible that this was also the first ster» with vitamin A esters. l$k The carbonium ion loses a proton from the cyclohexene ring to form the 6th double bond. This initial reaction could be considered an ioniza tion represented by a typical ionization equilibrium equation. R'Cocf OH' \ Keq The effects of the (R-COO” ) or (0H“ ) which are known to prevent elimination in these cases can easily be seen from the Law of Mass Action. Large quantities of these species can suppress ionization of vitamin A by a common ion effect. The second method of stabilization suggested is also possible since it is known that unsaturated compounds may undergo addition reactions with acids. Since Henbest (132) suggested that ethanol can add on the cyclohexene ring of anhydrovitamin A, it would also appear that this was the initial step in the addition reaction with fatty acids. This possibility was investigated further by treat ing anhydrovitamin A with "99%", "95%", "85$" isopropyl alcohol, cyclohexane and fatty acid solutions in the solvents mentioned. The isopropyl alcohol and hydroalcoholic solvents 155 reacted with the substrate to reform a vitamin A substance. Both fatty acids and water accelerated the rates of formation. Vitamin A esters were formed in cyclohexane fatty acid solutions, but the controls did not contain any vitamin A substance. Reaction Products Chromatographic analysis of these anhydrovitamin A systems showed that the isopropyl alcohol fatty acid solutions contained a mixture of vitamin A alcohol and ester as their "active" components. As the amount of water in these reaction mixtures was increased the ratio of vitamin A alcohol to ester also increased. The controls contained only the vitamin A alcohol. It was apparent that the larger amounts of vitamin A alcohol were related to the larger amounts of water present. It has been previously stated that vitamin A treated with base will not form anhydrovitamin A (56). The action of HC1 on vitamin A can be terminated by base, but anhydro vitamin A treated with base will not reform a vitamin A substance. Patty acids catalyze the formation of a vitamin A substance from anhydrovitamin A, which supports the second suggested method of stabilization of vitamin A, namely that the fatty acids react with anhydrovitamin A to reform a vitamin A substance and that the reaction is acid catalyzed. 156 Action of Mater The presence of water had a catalytic effect on both types of reactions studied (destruction and formation). Its effect appeared to be solely catalytic, hastening the approach of equilibrium regardless of the starting material .(vitamin A ester or ..anhydrovitamin A).°“ This catalytic ° c „0; action could be related to increased ionization of the .re actants due to the high dielectric constant of water. Other hydroxylated solvents as methanol, ethanol and isopropanol act by similar mechanisms but, due to lower polarity and solvation power, they are.less destructive. Oxidation and polymerization of anhydrovitamin A also force the reaction, to the right. . Two rates (k^ and k^) were present; in systems having "99%" isopropyl alcohol as the solvent, regardless of whether vitamin A or anhydrovitamin A was the substrate, k^ was always greater than k2 . - It appeared that k-j^ was related to the small water, content (0.056$) of the "99%"' •' isoproponal because the "95%" and "‘85%" systems only usually showed one rhte. _ : - . It would be logical to assume that k-^ in the -systems «. ® ® ,• containing the ester was a sum of 2 rates of decomposition; one due to the water of the media and the other due to the isopropyl alcohol. When the small quantity of water was consumed k£, which was slower, made its appearance. It has been shown that water had a catalytic effect on the re actions in both directions. k-j_ indicates a catalytic effect 157 when compared to k2 which is a further indication that may be related to the small concentration of water present. Mechanisms of Decomposition of the Esters and Vitamin A Alcohol Based on the assumption that decreased electronega tivity around the alkyl oxygen or carbonyl oxygen would reduce proton catalyzed attack, a series of esters were synthesized showing such properties. When the stability of the esters was determined in "99%" and "95$" isopropyl alcohol with and without fatty acids the order of stability was as follows: ^Vitamin A trichloroacetate •Hd cd Vitamin A chloroacetate •P W •H Vitamin A chloropropionate rH rH c!bo •H O •H od o ra Vitamin A acrylate •p o cd ra h o cd U Vitamin A crotonate bO o d i—| d • H |> a H m Pi Vitamin A sorbate cd o ® P P Pi Vitamin A acetate o o d ra I—I *H Vitamin A palmitate It appeared that there was a definite correlation between, the Ka of the acid radical of the ester and decreased stability against elimination in isopropyl alcohol. The trichloroacetate was least stable and the palmitate most stable. The 5 percent of water in the "95$" isopropyl alcohol system had a generalized catalytic action toward elimination. From these data, certain conclusions regarding the mechanism of decomposition may be reached. The 158 electronegative groups in the 2 position increased the Ka of the acid and simultaneously reduced the electron density around the carbonyl and alkyl oxygen. These factors weaken the covalent alkyl oxygen bond (X), rendering it more more susceptible to solvolysis in alcohol and water as shown with this series of esters. No anhydrovitamin A was formed in hydrocarbon solvents. When the vitamin A esters, alcohol, neo-alcohol and methyl ether were treated with HCl in anhydrous ethanol the order of stability was reversed. Vitamin A chloroacetate m Vitamin A alcohol'* G o O G Neovitamin A alcohol'* vs 1>5 Sll Vitamin A methyl ether”“' H 01 as •H I w fit H Vitamin A sorbate cd o bO -P « g m H m Vitamin A acetate 60g cd G © • h i d G Vitamin A palmitate GO G H o cd P O g © O G H Vitamin A crotonate G ft cd u fix! G O -P Vitamin A acrylate H O © VVitamin A chloropropionate v ‘“'These forms are not esters but are included for purposes of comparison. The reason for the position or vitamin A chloroacetate is difficult to explain except that the -C-0- bond may be so 159 weak that the sum of the rates of the two types of reactions simultaneously Is very large. The stability of the other esters varied directly with the Ka . When these compounds were tested for stability in HC1- ethanol USP they were rated as follows: Vitamin A chloroacetate J>a Vitamin A o( chloropropionate -p •H rH Vitamin A alcohol •P rO cd .p Vitamin A methyl ether CQ so Vitamin A acetate c * Cj •H m Vitamin A sorbate -„ - ca 0 5h o Vitamin A crotonate C! H Vitamin A palmitate vyvitamin A acrylate Since the o( chloropropionate was the most stable in HC1- anhydrous ethanol it appeared strange that it would show a large decrease in stability when water was added. This is probably due to the sensitivity to solvolysis of esters containing radicals with high Ka values. The water reduced the rate of elimination about 10 times when compared to the -anhydrous system. This decreased rate of elimination may be related" to the1 differences in basicity of water and ethanol. It is logical to assume that ethanol is a stronger base than water, thus it removes the proton from the cyclohexene ring with greater ease. The presence of the water in USP ethanol reduces the basicity of the media with a consequential de crease in the rate or elimination or the proton from the 160 cyclohexene ring. The reduced basicity is related to the competition of water molecules and the proton from the cyclohexene ring for the oxygen of ethanol. The stability of the esters in HCl-ethanol USP appeared to be a sum of rates of proton and solvolytic attack with the acrylate showing the best stability. . Ingold (iflj.) described a method of hydrogen attack on an ester with the formation of a carbonium ion. 0 (1 ) R j— C---0 — R + H Tast R---C - OHR 8 8 ; ' (2) B'— C - OHR slow",- ' ^R1 - C - 'OH■ + R- ■ The difference in stability of these compounds in solvents with and without acid presented evidence of two types of decomposition: 1. Proton Attack - It appeared that the alkyl oxygen was attacked by hydrogen ions. The esters containing acid radicals with the higher Ka were attacked the least due to the reduced electron density around the alkyl oxygen. Vitamin A alcohol was apparently very strongly attacked by acids. This could be expected since the electronegative effect of hydrogen was less than that of the ester group and the electron density around the alkyl oxygen of vitamin A alcohol was greater than the ester, favoring acid attack. 2. Solvent Attack - Under these conditions stability was dependent on the strength of the carbon oxygen bond. Vitamin A alcohol was known to be very stable in ethanol, 161 whereas the esters were less stable (5&). This could be explained since the C-0- bond of vitamin A alcohol was stronger and was not easily weakened by basic solvents. In the ester the covalent bond is weakened because electrons are drawn away by the ester group and the bond was more susceptible to solvent attack. Solvent attack is further substantiated by the work of Higuchi and Reinstein (5&) who showed that pyridine in ethyl alcohol had no effect on the rate of elimination of vitamin A acetate. Since the pyridine tied up protons the mechanism of decomposition was not proton attack and appeared to be due to the action of the ethanol on the ester. Stability by the one mechanism caused a correspond ing decrease in stability by the other mechanism. Therefore the structure of the ester should be a compromise. Vitamin A is rarely used in a strongly acid media ( p H £) thus it would be most desirable to synthesize a molecule which is more stable against solvolytic attack, than acid attack. It appeared that the acid radicals with very low K& values would be best suited to fill this need. The combined methods of decomposition are shown below: 4RC00H J + H C & ^ tyif&ooH ■ 7 162 SUMMARY AND CONCLUSIONS 1. The stability of vitamin A palmitate in a variety of ointment bases was studied. 2. Vitamin A palmitate in hydrocarbon solvents such as n-hexane showed only oxidative degradation, whereas vitamin A in an alcoholic solvent, such as isopropyl alcohol, showed both elimination and oxidation. 3. The presence of fatty acids in isopropyl alcohol solutions of vitamin A esters reduced the loss of the vitamin and maintained a lower concentration of anhydro vitamin A when compared to a control without fatty acids. i|. The addition of water to isopropyl alcohol increased the rate of vitamin A loss and anhydrovitamin A formation. Patty acids still maintained their stabilizing action. 5. The reverse reaction, with anhydrovitamin A plus isopropyl alcohol alone and with fatty acid showed that fatty acids accelerated vitamin A substance formation, when compared to isopropyl alcohol control. 6. The addition of water to the systems mentioned, in 5 accelerated vitamin A substance formation. The rates varied directly with temperature. 163 7. The reaction of anhydrovitamin A plus fatty acid in cyclohexane was found to be a second order reaction having a first order dependence on each reactant. 8. These systems were chromatographed on alumina and paper and the chromatographed fractions were identified by U.V. and I.R. spectra. 9. The stabilizing action of the fatty acid appeared to be cue to a catalytic regeneration of the ester in an hydrous solvents. A mixture of vitamin A ester and alcohol was found in hydroalcoholic solvents. 10. A method of synthesis for esters of vitamin A that are affected by water was developed. 11. A method of synthesis of sorbic acid chloride was developed. 12. The U.V. and I.R. spectra, Rf» values, , lcm saponification equivalents for the new esters were determined. 1 3 . The stability of these esters was determined in 1. alcoholic RaOH 2. Anhydrous ethanolic HC1 3. USP ethanolic-HCl I4.. "99%” and "95%" isopropyl alcohol 5. "99%” and "95%" isopropyl alcohol containing N/30 decanoic acid. 11|. 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Gillam, J., Norton, K. B., Rivett, D. E., Sutton, D. A., Biochem. J., t+3, 1+58 (1956). 181. Heilbron, I. M., Gillam, A. E., Morton, R. A., Biochem. J., 2£, 1352 (1931). 182. Gillam, A. E., Heilbron. I. M., Jones, W. E., Lederer,E., Ibid. 22, It-05 (1938). 1 8 3. Hamlet, J. C., Farrar, K. R., Henbest, H. B., Jones, E., J. Chem. Soc., 2657 (1952). m AUTOBIOGRAPHY I, Albert Joseph Forlano, was born In Brooklyn, New York, on August 18, 1929. I received my elementary education in New York public schools. I received my Bachelor of Science cum laude degree from the Brooklyn College of Pharmacy, Long Island University, on June 6, 1950* In June of 1951 I was inducted into the United States Army and spent the major part of my time in the Medical Corps. In June, 1953* I was honorably discharged from the Army. I began training for the Master of Science degree at Columbia University, College of Pharmacy, in September of 1953* which I received in June of 1955 I then went to work for Hoffmann La Roche in Nutley, New Jersey, for a period of six months at which time I de cided to return to school for my doctorate degree. In January of 1956 I began my doctorate training at the Ohio State University, College of Pharmacy. In September of 1956 I was appointed a Fellow of the American Foundation for Pharmaceutical Education at Ohio State University. I remained a fellow for three years while I completed my requirements for the degree of Doctor of Philosophy.