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Distribution and antimicrobial activity of preservatives in solubilized and emulsified systems Kazmi, Syed Jamshed Ali 1974

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DISTRIBUTION AND ANTIMICROBIAL ACTIVITY OF PRESERVATIVES IN SOLUBILIZED AND EMULSIFIED SYSTEMS by SYED JAMSHED ALI KAZMI B . S c , Agra U n i v e r s i t y , I n d i a , 1964 B.Pharm., U n i v e r s i t y of Ka r a c h i , P a k i s t a n , 1968 M.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the D i v i s i o n of Pharmaceutics of the Fa c u l t y of Pharmaceutical Sciences We accept t h i s t h e s i s as conforming to the r e q u i r e d standard: THE UNIVERSITY OF BRITISH COLUMBIA APRIL, 1974 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date M ^ i 28/1 X ABSTRACT U n t i l r e c e n t l y , e v a l u a t i o n of the e f f e c t i v e n e s s of pr e s e r v a t i v e s i n s o l u b i l i z e d and e m u l s i f i e d systems has depended l a r g e l y on time-consuming m i c r o b i o l o g i c a l techniques. Mathematical models have now been developed which enable the amount of p r e s e r v a t i v e necessary f o r adequate p r e s e r v a t i o n to be c a l c u l a t e d . These mathematical models have been derived on the assumption that the a n t i m i c r o b i a l a c t i v i t y of preser -v a t i v e i n s o l u b i l i z e d and e m u l s i f i e d systems i s l a r g e l y a f u n c t i o n of the f r e e p r e s e r v a t i v e c o n c e n t r a t i o n i n the aqueous phase. In t h i s work, a physicochemical and m i c r o b i o l o g i c a l e v a l u a t i o n was made tp t e s t the v a l i d i t y of the above assumption and to study the p o s s i b l e e f f e c t s of changes i n s u r f a c t a n t c o n c e n t r a t i o n and o i l - w a t e r r a t i o on the a n t i m i c r o -b i a l a c t i v i t y of p r e s e r v a t i v e i n s o l u b i l i z e d and e m u l s i f i e d systems. Some weaknesses of the e a r l i e r models were pointed out. In p a r t i c u l a r , the concept of the cap a c i t y of the system to r e s i s t the changes i n e f f e c t i v e f r e e p r e s e r v a t i v e c o n c e n t r a t i o n was developed. The p e r m e a b i l i t y of cellophane membranes to the nonionic s u r f a c t a n t cetomacrogol was i n v e s t i g a t e d using e q u i l i b r i u m d i a l y s i s , dynamic d i a l y s i s , and an u l t r a f i l t r a t i o n technique. Cellophane and s i l i c o n e rubber membranes were compared i n an e q u i l i b r i u m d i a l y s i s study of the i n t e r a c t i o n of c h l o r o c r e s o l w i t h nonionic s u r f a c t a n t s . The magnitude of e r r o r s introduced i n t o the bi n d i n g parameters usin g cellophane membrane were r e l a t e d to the p e r m e a b i l i t y of the membranes to the nonionic s u r f a c t a n t s and to changes i n volume and i n s u r f a c t a n t c o n c e n t r a t i o n which occurred" as a r e s u l t of osmotic d i f f e r e n t i a l across i i the membrane. The d i a f i l t r a t i o n technique was evaluated f o r the i n t e r a c t i o n of benzoic a c i d w i t h cetomacrogol. The r e s u l t s of the d i a f i l t r a t i o n technique were compared w i t h those obtained using the e q u i l i b r i u m d i a l y s i s technique. Various t e c h n i c a l a r t i f a c t s of the d i a f i l t r a t i o n technique were pointed out. I n t e r a c t i o n of p r e s e r v a t i v e mixtures w i t h the nonionic s u r f a c t a n t cetomacrogol was studied u s i n g the e q u i l i b r i u m d i a l y s i s technique. Attempts to c o r r e l a t e the data w i t h theory of competitive p r o t e i n b i n d i n g were un s u c c e s s f u l . The i n t e r a c t i o n of c h l o r o c r e s o l w i t h c e r t a i n n o n i o n i c s u r f a c t a n t s and t h e i r mixtures was stud i e d using the e q u i l i b r i u m d i a l y s i s technique. Binding parameters which c h a r a c t e r i z e d the i n t e r a c t i o n of the p r e s e r v a t i v e w i t h each i n d i v i d u a l s u r f a c t a n t were used to p r e d i c t the b i n d i n g behaviour of s u r f a c t a n t mixtures. Various f a c t o r s a f f e c t i n g the d i s t r i b u t i o n of p r e s e r v a t i v e s between o i l .and water and the i n t e r a c t i o n between p r e s e r v a t i v e s and s u r f a c t -ants are discussed. These f a c t o r s were r e l a t e d to the problem of the d i s t r i b u t i o n of a p r e s e r v a t i v e i n o i l and water emulsion systems. Methodology used to evaluate the v a r i o u s physicochemical parameters and the a n t i m i c r o b i a l a c t i v i t y i s reviewed, and equations f o r rep r e s e n t i n g the r e s u l t s are discussed. The M i l l i p o r e f i l t r a t i o n method was compared w i t h a pour-plate technique f o r the v i a b l e counting of E. e o l i . i i i The b a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n aqueous cetomacro-g o l s o l u t i o n s and l i q u i d p a r a f f i n emulsions of v a r y i n g o i l - w a t e r r a t i o s against c o l i was studied using a v i a b l e count method. The micro-b i o l o g i c a l r e s u l t s were r e l a t e d w i t h the physicochemical models of p r e s e r v a t i v e d i s t r i b u t i o n i n s o l u b i l i z e d and e m u l s i f i e d systems. This a b s t r a c t represents the true contents of the t h e s i s submitted. Signatures of Examiners: i v TABLE OF CONTENTS Page INTRODUCTION • 1 LITERATURE SURVEY 6 A. D i s t r i b u t i o n of P r e s e r v a t i v e s i n Oil-Water Systems . . . 6 (a) Factors a f f e c t i n g a n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s i n o i l - w a t e r systems . . 6 (b) Representation of d i s t r i b u t i o n data 12 B. I n t e r a c t i o n of P r e s e r v a t i v e s w i t h Nonionic Surfactants . 19 (a) P o t e n t i a t i o n of p r e s e r v a t i v e a c t i v i t y . . . . . . 19 (b) I n a c t i v a t i o n of p r e s e r v a t i v e a c t i v i t y 20 (c) Mechanism of i n a c t i v a t i o n . . . 22 (d) P o s s i b l e s i t e s f o r the i n t e r a c t i o n of p r e s e r v a t i v e s i n a s u r f a c t a n t m i c e l l e 24 (e) E f f e c t of p r e s e r v a t i v e s on the m i c e l l a r molecular weight of : s u r f a c t a n t s 27 ( f ) Representation of i n t e r a c t i o n data , 30 C. I n t e r a c t i o n of P r e s e r v a t i v e Mixtures w i t h Nonionic Sur f a c t a n t s . 33 D. Theory of Competitive Binding of Drugs w i t h Macromolecules . . .... . . ;. . . . . 34 E. I n t e r a c t i o n of a P r e s e r v a t i v e w i t h Mixtures of Nonionic Surfactants . 36 F. Representation of I n t e r a c t i o n Data f o r the Binding of a P r e s e r v a t i v e w i t h Mixtures of Nonionic Surfactants . . . 37 G. D i s t r i b u t i o n and A n t i m i c r o b i a l A c t i v i t y of P r e s e r v a t i v e s i n E m u l s i f i e d Systems . . 38 (a) I n t r o d u c t i o n 38 (b) C a l c u l a t i o n of t o t a l p r e s e r v a t i v e c o n c e n t r a t i o n r e q u i r e d i n an emulsion ^ TABLE OF CONTENTS (Continued) P H. Methodology (a) D i s t r i b u t i o n of p r e s e r v a t i v e s i n o i l - w a t e r systems . . (b) I n t e r a c t i o n of p r e s e r v a t i v e s w i t h n o n i o n i c s u r f a c t a n t s . . . . . . . . . . 17. Physico-chemical methods I I . B i o l o g i c a l methods . . . . (c) D i s t r i b u t i o n of p r e s e r v a t i v e s i n o,il-water-surfactant systems (d) A n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s i n , o i l - w a t e r -s u r f a c t a n t systems . EXPERIMENTAL ;  A. Apparatus B. M a t e r i a l s C. Temperature D. A n a l y s i s of Surface-Active Agents . . . . E. A n a l y s i s of P r e s e r v a t i v e s i n Aqueous and Surfactant S o l u t i o n s F. A n a l y s i s of P r e s e r v a t i v e Mixtures i n Aqueous and Surfactant S o l u t i o n s . . . . G. P e r m e a b i l i t y of Membranes to Nonionic Surfactants . . . . H. Binding of C h l o r o c r e s o l w i t h S i l i c o n e Rubber i n E q u i l i b r i u m D i a l y s i s . I . I n t e r a c t i o n of P r e s e r v a t i v e s w i t h Nonionic Surfactants . . J . I n t e r a c t i o n of P r e s e r v a t i v e Mixtures w i t h Cetomacrogol . . K. I n t e r a c t i o n of C h l o r o c r e s o l w i t h Mixtures of Nonionic Surfactants L. D i s t r i b u t i o n of C h l o r o c r e s o l i n L i q u i d Paraffin-Water Systems . . . . . . vx TABLE OF CONTENTS (Continued) Page M. D i s t r i b u t i o n of C h l o r o c r e s o l i n L i q u i d P a r a f f i n -Water-Cetomacrogol Systems 93 N. M i c r o b i o l o g i c a l Procedures 94 (a (b (c (d: (e (f (g (h ( i (j (k (1 Organism 94 C u l t u r e media 94 S t e r i l i z a t i o n of experimental s o l u t i o n s . . . . S t e r i l i z a t i o n of m i l l i p o r e f i l t r a t i o n equipment . B a c t e r i a l c u l t u r e s C o n s t r u c t i o n of standard curve f o r E. c o l i . . . 94 95 95 95 95 97 97 B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n water . . 98 B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n cetomacrogol s o l u t i o n s 98 B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n l i q u i d -P r e p a r a t i o n of standard E.. c o l i suspension . . V i a b l e count method using pour-plate technique V i a b l e count method using membrane f i l t r a t i o n technique . . . . . . . . . . p a r a f f i n emulsions 99 RESULTS AND DISCUSSION A. P e r m e a b i l i t y of Membranes to Nonionic Surfactants . . . B. Volume Change due .to Osmosis i n D i a l y s i s Studies . . . C. I n t e r a c t i o n of P r e s e r v a t i v e s w i t h Nonionic Su r f a c t a n t s . (a) E q u i l i b r i u m d i a l y s i s technique (b) D i a f i l t r a t i o n technique D. I n t e r a c t i o n of P r e s e r v a t i v e Mixtures w i t h Cetomacrogol . 100 100 100 103 103 111 V1X TABLE OF CONTENTS (Continued) Page E. I n t e r a c t i o n of C h l o r o c r e s o l w i t h Mixtures of Some Nonionic Surfactants . . . . F. D i s t r i b u t i o n of C h l o r o c r e s o l i n L i q u i d P a r a f f i n -T, 141 Water Systems G. C a l c u l a t i o n of Required C h l o r o c r e s o l Concentration i n Cetomacrogol S o l u t i o n s H. C a l c u l a t i o n of Required C h l o r o c r e s o l Concentration i n L i q u i d P a r a f f i n Emulsions I . C o r r e l a t i o n of Physico-Chemical Data w i t h A n t i m i c r o b i a l A c t i v i t y 1 4 6 (a) Comparison between m i l l i p o r e f i l t r a t i o n and pour-plate techniques . (b) B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n water . . . ^ ® (c) B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n cetomacrogol s o l u t i o n s . 1^6 (d) B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n l i q u i d p a r a f f i n emulsions 170 SUMMARY . 1 8 8 REFERENCES APPENDICES 193 206 v i i i Table 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. LIST OF TABLES Page Pro p o r t i o n s of benzoic a c i d u n d i s s o c i a t e d at v a r i o u s pH values 7 Influence of p a r t i t i o n c o e f f i c i e n t and phase-volume r a t i o n o n c o n c e n t r a t i o n of p r e s e r v a t i v e i n aqueous and o i l phase of a two-phase system 10 Volume change due to osmosis i n e q u i l i b r i u m d i a l y s i s using F i s h e r cellophane as a semipermeable membrane .. , 104 Comparison of ' r ' f o r a given t o t a l amount of c h l o r o c r e s o l i n e q u i l i b r i u m d i a l y s i s using s i l i c o n e rubber and F i s h e r cellophane as semipermeable membranes .105 Change of s u r f a c t a n t c o n c e n t r a t i o n i n e q u i l i b r i u m d i a l y s i s w i t h cellophane as a semipermeable membrane - - 109 C h l o r o c r e s o l c o n c e n t r a t i o n i n l i q u i d - p a r a f f i n ' emulsions s t a b i l i z e d w i t h cetomacrogol . 148 Comparison of slopes i n F i g . 42 using ' t ' t e s t 159 Comparison between Eq. 28 and 62 f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol 169 Comparison of slopes i n F i g . 45 using ' t ' t e s t 172 Computer program f o r the n o n l i n e a r r e g r e s s i o n a n a l y s i s of the b i n d i n g data 208 i x LIST OF FIGURES Figure Page 1. P o s s i b l e s i t e s of i n c o r p o r a t i o n of s o l u b i l i z a t e s i n a m i c e l l e 25 2. V a r i a t i o n of m i c e l l a r molecular weight w i t h degree of s a t u r a t i o n of cetomacrogol m i c e l l e w i t h v a r i o u s s o l u t e s 29 3. E q u i l i b r i u m u l t r a f i l t r a t i o n apparatus 53 4. Polarographic current v o l t a g e curves 74 5. C a l i b r a t i o n curve f o r the polarographic determination of cetomacrogol 75 6. Spectrophotometric curves f o r benzoic a c i d and s o r b i c a c i d i n 0.01 N HCl . . • • • 79 7. Q curve f o r benzoic and s o r b i c a c i d s 80 8. Spectrophotometric curves f o r c h l o r o c r e s o l and methyl paraben i n water . 81 9. Q curve f o r c h l o r o c r e s o l and methyl paraben 82 10. Spectrophotometric curves f o r c h l o r o c r e s o l and pr o p y l paraben 83 11. Q curve f o r c h l o r o c r e s o l and p r o p y l paraben 84 12. Spectrophotometric curves f o r c h l o r o x y l e n o l and methyl paraben . . : 85 13. Q curve f o r c h l o r o x y l e n o l and methyl paraben . . . . 86 i . 14. Standard curve f o r E_. c o l i using, spectrophotometric technique 96 i 15. P e r m e a b i l i t y of cellophane membranes to cetomacrogol i n e q u i l i b r i u m d i a l y s i s 101 16. P e r m e a b i l i t y of F i s h e r cellophane membrane to cetomacrogol i n dynamic d i a l y s i s under s i n k c o n d i t i o n s . . . 102 17. Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol 107 LIST OF FIGURES (Continued) Page Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol HO Scatchard p l o t f o r the i n t e r a c t i o n of benzoic a c i d w i t h cetomacrogol . . 112 Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol i n absence and presence of methyl paraben 119 Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol i n absence and presence of p r o p y l paraben 120 Scatchard p l o t f o r the i n t e r a c t i o n of p r o p y l paraben w i t h cetomacrogol 121 Scatchard p l o t f o r the i n t e r a c t i o n of methyl paraben w i t h cetomacrogol i n absence and presence of c h l o r o c r e s o l 123 Scatchard p l o t f o r the i n t e r a c t i o n of methyl paraben w i t h cetomacrogol i n absence and presence of c h l o r o x y l e n o l 124 Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o x y l e n o l w i t h cetomacrogol . 125 Scatchard p l o t f o r the i n t e r a c t i o n of benzoic a c i d w i t h cetomacrogol i n absence and presence of s o r b i c a c i d . 126 Scatchard p l o t f o r the i n t e r a c t i o n of s o r b i c a c i d w i t h cetomacrogol . . 127 Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h Texofor A16, Texofor A60 and mixtures of Texofor A16 and A60 I 3 2 Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol, polysorbate 80 and mixtures of cetomacrogol and polysorbate 80 134 V a r i a t i o n of f r e e p r e s e r v a t i v e c o n c e n t r a t i o n w i t h t o t a l p r e s e r v a t i v e c o n c e n t r a t i o n f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h mixtures of Texofor A16 and Texofor A60 . 136 LIST OF FIGURES (Continued) Figure Page 31. Variation of free preservative concentration with total preservative concentration for the interaction of chlorocresol with mixtures of cetomacrogol and polysorbate 80 137 32. I-Dt"^  v e r s u s ^LB a t constant [D^] for the interaction of chlorocresol with Texofor A 16, Texofor A60 and mixtures of Texofor A16 and Texofor A60 139 33. l-^t^ v e r s u s HLB at constant [ D ^ j f orttheiinteraction of chlorocresol with cetomacrogol, polysorbate 80, and mixtures of cetomacrogol and polysorbate 80 140 34. Concentration of preservative in o i l as a function of free preservative in the aqueous phase for the distribution of chlorocresol between liquid paraffin and water 142 35,. Variation of free preservative concentration with total preservative concentration for the interaction of chlorocresol with cetomacrogol 144 36. Binding of.chlorocresol with silicone membrane 145 37. Variation of free chlorocresol concentration in the aqueous phase of the emulsion, with total chlorocresol for an 0/W emulsion containing 50% v/v liquid paraffin emulsified with 3% w/v cetomacrogol 147 38. Comparison between Millipore f i l t r a t i o n and pour-plate techniques for the enumeration of _E. c o l i . , 151 39. Semilogarithmic plot of the number of organisms/ml as a function of time for the survival of E_. c o l i in water, aqueous cetomacrogol solutions and liquid paraffin emulsions . . 152 40. Semilogarithmic plot of the number of organisms/ml as a function of time for the bactericidal activity of chlorocresol in water against IS. c o l i . . . . . . . . * • 153 41i. Probit % survivors as a function of time for the bactericidal activity of chlorocresol in water against E. c o l i . . ..; . 1 5 4 x i i LIST OF FIGURES (Continued) Figure Page 42. Probit % survivors as a function of time for the bactericidal activity of chlorocresol in aqueous cetomacrogol solutions against IS. c o l i 157 43. Comparison between percent loss of chlorocresol from water and"aqueous phase of cetomacrogol when equal amount of preservative i s removed from water and cetomacrogol solution . . . . . 161 44. Capacity as a function of surfactant concentration for the interaction of chlorocresol with Texofor A16, Texofor A23 (cetomacrogol) and Texofor A60 163 45. Probit % survivors of E. c o l i as a function of time for the bactericidal activity of chlorocresol in water and liquid paraffin emulsions stabilized with cetomacrogol : 171 46. Comparison between percent loss of chlorocresol from water and aqueous phase of an oil-water dispersion when equal amount of preservative i s removed from water and the oil-water dispersion . . . 177 47. Capacity as a function of oil-water partition coefficient for the distribution of chlorocresol in oil-water dispersions 179 48. Capacity as a function of oil-water ratio for the distribution of chlorocresol in oil-water dispersions . 180 49. Comparison between percent loss of chlorocresol from water and aqueous phase of an oil-water emulsion stabilized with cetomacrogol when equal amounts of preservative are removed from water and the emulsion . 181 50. Capacity as a function of oil-water ratio for the distribution of chlorocresol in oil-water emulsions stabilized with cetomacrogol . . . 183 51. Capacity as a function of oil-water ratio for the distribution of chlorocresol in liquid-paraffin emulsions stabilized; with cetomacrogol 184 x i i i LIST OF FIGURES (Continued) Figure Page 52. Capacity as a f u n c t i o n of o i l - w a t e r r a t i o f o r the d i s t r i b u t i o n of c h l o r o c r e s o l i n o i l - w a t e r emulsions s t a b i l i z e d w i t h cetomacrogol 185 53. A n a l y s i s of antagonism using Scatchard p l o t 220 x i v ACKNOWLEDGEMENTS I wish to express my s i n c e r e g r a t i t u d e and a p p r e c i a t i o n t o : Dr. A.G. M i t c h e l l f o r h i s encouragement and guidance during the course of t h i s i n v e s t i g a t i o n . Dr. J.E. Axelson, Dr. M. Pernarowski and Dr. B.D. Roufogalis f o r many v a l u a b l e , i n f o r m a t i v e and h e l p f u l d i s c u s s i o n s . Dr. B.C. McBride (Department of Micr o b i o l o g y ) f o r c o u n s e l l i n g i n the m i c r o b i o l o g i c a l aspects of t h i s study. Dr. F.P. G l i c k (Department of Mathematics) f o r advice i n the s t a t i s t i c a l a n a l y s i s of m i c r o b i o l o g i c a l data. Dr. M.H. Chaudhry (IPEC L t d . ) , Dr. A.W. Khanzada (Department of Chemistry), Mr. A.K. Khatry (Department of Mechanical Engineering) and Mrs. P. Haugen f o r t h e i r v a l u a b l e a s s i s t a n c e i n preparing computer programs. F i n a n c i a l support from the U n i v e r s i t y of B r i t i s h Columbia i s g r a t e f u l l y acknowledged. DEDICATION To my parents 1 INTRODUCTION Modern usage of the word s o l u b i l i z a t i o n appears to have given i t a more general meaning and, t h e r e f o r e , i t would perhaps r e q u i r e a s p e c i f i c d e f i n i t i o n f o r the present work. S o l u b i l i z a t i o n can be defined as the p r e p a r a t i o n of a thermodynamically s t a b l e i s o t r o p i c s o l u t i o n of a substance normally i n s o l u b l e or very s l i g h t l y s o l u b l e i n water by the i n t r o d u c t i o n of a s u r f a c t a n t or a mixture of s u r f a c t a n t s . Thus, a s o l u b i l i z e d system means a system c o n t a i n i n g a p r e s e r v a t i v e or a mixture of p r e s e r v a t i v e s and one or more types of s u r f a c t a n t s . For the present purposes an e m u l s i f i e d system i s defined as a heterogeneous o i l - i n - w a t e r d i s p e r s i o n s t a b i l i z e d by a s u r f a c t a n t . The s u r f a c t a n t s used i n t h i s work were of the nonionic type. The m i c r o b i o l o g i c a l a c t i v i t y of a p r e s e r v a t i v e i n s o l u b i l i z e d and e m u l s i f i e d systems ismmuch more complex than i n simple aqueous systems but i s g e n e r a l l y l e s s than the same amount of p r e s e r v a t i v e i n an aqueous system. An understanding of the f a c t o r s c o n t r o l l i n g the e f f e c t i v e n e s s of a p r e s e r v a t i v e i n these systems can only be achieved by a thorough study of the v a r i o u s p h y s i c a l , chemical, and m i c r o b i o l o g i c a l parameters governing the d i s t r i b u t i o n and the a n t i m i c r o b i a l a c t i v i t y . At the present time, assessment of the a b i l i t y of p r e s e r v a t i v e s to prevent m i c r o b i a l s p o i l a g e of s o l u b i l i z e d and e m u l s i f i e d products depends l a r g e l y on e m p i r i c a l t e s t s i n v o l v i n g i n o c u l a t i o n of the f i n i s h e d product, and examination during a prolonged p e r i o d of storage. These methods are l a b o r i o u s , time consuming and are mainly q u a l i t a t i v e i n nature. No 2 informati o n i s obtained w i t h regard to the mechanism of i n a c t i v a t i o n of the p r e s e r v a t i v e or the conc e n t r a t i o n of p r e s e r v a t i v e i n v a r i o u s phases of the s u r f a c t a n t s o l u t i o n or emulsion. Mathematical models have now been developed which permit a p r e d i c t i o n to be made of the p r e s e r v a t i v e concentration r e q u i r e d i n a s o l u b i l i z e d system or an emulsion i n order to achieve adequate p r e s e r v a t i o n . The development of these models has been based on the b a s i c assumptions that the a n t i m i c r o b i a l a c t i v i t y i s l a r g e l y a f u n c t i o n of the conc e n t r a t i o n of p r e s e r v a t i v e i n the aqueous phase, and that the p r e s e r v a t i v e p a r t i t i o n e d i n t o the o i l phase or bound w i t h the s u r f a c t a n t m i c e l l e s i s b i o l o g i c a l l y i n a c t i v e . In t h i s work a physico-chemical and m i c r o b i o l o g i c a l e v a l u a t i o n of two mathematical models, one f o r s o l u b i l i z e d systems and the other f o r emulsions, has been made to t e s t the v a l i d i t y of the above assumptions, and to study the i n f l u e n c e of f a c t o r s such as s u r f a c t a n t c o n c e n t r a t i o n and o i l - w a t e r r a t i o on the a n t i m i c r o b i a l a c t i v i t y . The a n t i m i c r o b i a l a c t i v i t y of a p r e s e r v a t i v e i n a system i s governed by the a v a i l a b i l i t y of the e f f e c t i v e f r e e p r e s e r v a t i v e c o n c e n t r a t i o n to the micro-organisms. D e p l e t i o n of the p r e s e r v a t i v e due to f a c t o r s such as i n t e r a c t i o n w i t h micro-organisms or f o r e i g n m a t e r i a l s , chemical decompo-s i t i o n , or even metabolism by the micro-organisms, w i l l reduce the f r e e p r e s e r v a t i v e c o n c e n t r a t i o n and hence the a n t i m i c r o b i a l a c t i v i t y . The d u r a t i o n of a c t i v i t y w i l l depend on the ca p a c i t y of the system. Capacity i s defined as the a b i l i t y of the system to r e s i s t changes i n the e f f e c t i v e f r e e p r e s e r v a t i v e c o n c e n t r a t i o n . In aqueous s o l u t i o n s the a c t i v i t y decreases r a p i d l y because these systems l a c k c a p a c i t y . In s o l u b i l i z e d and e m u l s i f i e d 3 systems the s u r f a c t a n t m i c e l l e s and the o i l phase-act as r e s e r v o i r s of p r e s e r v a t i v e and hence, c a p a c i t y i s c o n t r o l l e d by the s u r f a c t a n t concen-t r a t i o n , the o i l - w a t e r p a r t i t i o n c o e f f i c i e n t and the o i l - w a t e r r a t i o . Hence, the o v e r a l l e f f e c t i v e n e s s of the p r e s e r v a t i v e i s determined not only by the e f f e c t i v e f r e e p r e s e r v a t i v e c o n c e n t r a t i o n b u t . a l s o by the c a p a c i t y of the system. In the present work, an attempt has been made to develop a j numerical expression f o r the ca p a c i t y and to examine the ./relationship between ca p a c i t y and the parameters such as s u r f a c t a n t c o n c e n t r a t i o n , o i l - w a t e r p a r t i t i o n c o e f f i c i e n t and o i l - w a t e r r a t i o f o r v a r i o u s s o l u b i l i z e d and emulsif.ied systems. The use of mathematical models to c a l c u l a t e the t o t a l c o n c e n t r a t i o n of p r e s e r v a t i v e necessary to achieve the de s i r e d c o n c e n t r a t i o n i n the aqueous phase of a s u r f a c t a n t s o l u t i o n or an emulsion r e q u i r e s the determination of va r i o u s physico-chemical parameters. Methods f o r determining the d i s t r i b u t i o n of p r e s e r v a t i v e between o i l - w a t e r phases are w e l l e s t a b l i s h e d . The i n t e r a c t i o n between p r e s e r v a t i v e s and s u r f a c t a n t s has been studied using a v a r i e t y of methods, e.g., e q u i l i b r i u m d i a l y s i s , dynamic d i a l y s i s , g e l f i l t r a t i o n , e t c . D i a l y s i s methods are slow and r e q u i r e numerous experiments to f u l l y c h a r a c t e r i z e the i n t e r a c t i o n . The s e l e c t i o n of an adequate semipermeable membrane i s a very c r u c i a l aspect of d i a l y s i s methodology. I d e a l l y d i a l y s i s i n v e s t i g a t i o n s r e q u i r e that the membrane be impermeable to the nonio n i c s u r f a c t a n t w h i l e a l l o w i n g d i f f u s i o n of the s o l u t e and that the osmotic d i f f e r e n t i a l across the membrane i s n e g l i g i b l e . Cellophane membranes are w i d e l y used i n e q u i l i b r i u m and dynamic d i a l y s i s s t u d i e s i n v o l v i n g i n t e r -a c t i o n of p r e s e r v a t i v e s and drugs, w i t h nonionic s u r f a c t a n t s . Cellophane has a l s o been used to study the e f f e c t s of s u r f a c t a n t s on the d i f f u s i o n of 4 drugs across.the membranes. There i s some controversy concerning the p e r m e a b i l i t y of cellophane d i a l y s i s membranes to nonionic s u r f a c t a n t s . Hence, i n view of the continued use of cellophane, q u a n t i t a t i v e measure-ments of the p e r m e a b i l i t y of cellophane membranes to nonionic s u r f a c t a n t s , and an assessment of the e f f e c t s of s u r f a c t a n t permeation and osmosis on the b i n d i n g constants f o r the i n t e r a c t i o n between a p r e s e r v a t i v e and nonionic s u r f a c t a n t s has been made i n t h i s study. Because of the d i f f i c u l t i e s i n d i a l y s i s methods i t i s r e a l i z e d that there i s s t i l l need f o r a simple, r a p i d and r e l i a b l e means of e v a l u a t i n g the bi n d i n g parameters. The d i a f i l t r a t i o n technique appeared to answer these needs. Therefore, the d i a f i l t r a t i o n technique was used i n an attempt to determine b i n d i n g para-meters f o r the p r e s e r v a t i v e - s u r f a c t a n t i n t e r a c t i o n . While the i n t e r a c t i o n of p r e s e r v a t i v e s w i t h nonionic s u r f a c t a n t s has been studied e x t e n s i v e l y , l i t t l e i n f o r m a t i o n i s a v a i l a b l e concerning the s o l u b i l i z a t i o n of p r e s e r v a t i v e mixtures by nonionic s u r f a c t a n t s . Since p r e s e r v a t i v e mixtures are o f t e n employed i n cosmetic and pharmaceutical preparations c o n t a i n i n g nonionic s u r f a c t a n t s , an attempt has been made to examine the b i n d i n g behavior of b i n a r y p r e s e r v a t i v e mixtures w i t h a nonionic s u r f a c t a n t , and to c o r r e l a t e the bi n d i n g r e s u l t s w i t h the theory of competitive p r o t e i n b i n d i n g . Surfactant mixtures o f f e r many advantages over i n d i v i d u a l s u r f a c t a n t s and are e x t e n s i v e l y used t h e r e f o r e i n the fo r m u l a t i o n of s o l u b i l i z e d and e m u l s i f i e d systems. Hence, an attempt has been made to use bi n d i n g constants c h a r a c t e r i z i n g the i n t e r a c t i o n of a p r e s e r v a t i v e w i t h i n d i v i d u a l s u r f a c t a n t s to p r e d i c t the bi n d i n g behavior of s u r f a c t a n t mixtures. 5 Pour-plate technique or r o l l tube method are g e n e r a l l y used f o r the v i a b l e counting of micro-organisms. These techniques i n v o l v e d i l u t i o n of the sample i n some non-nutrient medium, such as normal s a l i n e or quarter s t r e n g t h Ringer's s o l u t i o n , and subsequent p l a t i n g on the agar. A few d i f f i c u l t i e s a r i s e w i t h the use of these techniques f o r studying the death r a t e of micro-organisms i n systems c o n t a i n i n g p r e s e r v a t i v e s . Large d i l u t i o n s are r e q u i r e d to reduce the co n c e n t r a t i o n of p r e s e r v a t i v e which would other-, wise i n h i b i t the growth of micro-organisms upon p l a t i n g . This i s a se r i o u s l i m i t a t i o n , e s p e c i a l l y when the death r a t e i s followed up to 100% m o r t a l i t y . Since at high m o r t a l i t y l e v e l s l a r g e d i l u t i o n s are not p o s s i b l e , the chances of carry-over of p r e s e r v a t i v e to the growth.medium are great. Recently membrane f i l t r a t i o n methods have been used e x t e n s i v e l y f o r the v i a b l e counting of micro-organisms. These methods are s a i d to obviate the aforementioned problems of the pour-plate or r o l l tube methods by r i n s i n g the t e s t f i l t e r w i t h j s t e r i l e f l u i d a f t e r sample f i l t r a t i o n . Hence, i n the present work the M i l l i p o r e f i l t r a t i o n method has been evaluated f o r the v i a b l e counting of 15. c o l i . 6 LITERATURE SURVEY A. D i s t r i b u t i o n of P r e s e r v a t i v e s i n Oil-Water Systems (a) Factors a f f e c t i n g the a n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s i n o i l - w a t e r systems A,p r e s e r v a t i v e added to an o i l - w a t e r mixture p a r t i t i o n s between the two phases (Hibbott and Monks, 1961; Bean, Richard and Thomas, 1962). The a n t i m i c r o b i a l a c t i v i t y i s mainly a f u n c t i o n of the a v a i l a b i l i t y , or thermodynamic a c t i v i t y , of a b i o l o g i c a l l y e f f e c t i v e c o n c e n t r a t i o n of preser -v a t i v e i n the aqueous phase, and not the t o t a l amount added, i . e . , p r e s e r v a t i v e i n the o i l phase i s b i o l o g i c a l l y i n a c t i v e (Wolffhugel and Von Knorre, 1881; C l a r k , 1939, Gershenfeld and B r i l l h a r t , 1939; A t k i n s , 1950; Bean, Richard and Thomas, 1962; Bean and Heman-Ackah, 1964; Bean, Heman-Ackah and Thomas, 1965). The a v a i l a b i l i t y of p r e s e r v a t i v e i n the aqueous phase i s c o n t r o l l e d by v a r i o u s f a c t o r s : 1. D i s s o c i a t i o n constant (K &) of the p r e s e r v a t i v e and the pH of the aqueous phase. 2. Oil-water p a r t i t i o n c o e f f i c i e n t (K°). ' w 3. Oil-water r a t i o ( q ) . 4. I n t e r f a c i a l f a c t o r . 5. Temperature. 6. E f f e c t of a d d i t i v e s . (1) D i s s o c i a t i o n constant of p r e s e r v a t i v e and pH of aqueous phase: Weak a c i d p r e s e r v a t i v e s are g e n e r a l l y most e f f e c t i v e i n t h e i r u n d i s s o c i a t e d form (Rahn and Conn, 1940; B a n d e l i n , 1958), the e q u i l i b r i u m between 7 u n d i s s o c i a t e d a c i d and anion being a f u n c t i o n of pH. I t can be observed from Table 1 that 60 times as much benzoic a c i d i s re q u i r e d at pH 6 as at pH 3 to achieve equivalent a n t i m i c r o b i a l a c t i v i t y . Table 1 P r o p o r t i o n s of Benzoic A c i d Undissociated at Various pH Values (Kostenbauder, 1962) pH Undissociated Benzoic A c i d % 2 99.4 3 94.3 4 62.5 5 13.7 6 1.6 K = 6.3 x 10"5 a pK = 4.2 a I f the minimum inhibitory Concentration of u n d i s s o c i a t e d a c i d i s known, the t o t a l c o n c e n t r a t i o n of a c i d r e q u i r e d i n the aqueous phase can be c a l c u l a t e d from the f o l l o w i n g equation (Kostenbauder, 1962): p I n h i b i t o r y Concentration - i r ^ q + ^ „ - , of un d i s s o c i a t e d a c i d ' a T o t a l r e q u i r e d p r e s e r v a t i v e = , [K 3 A c l o s e r e l a t i o n i s g e n e r a l l y found between pH and the a n t i -m i c r o b i a l a c t i v i t y of weak a c i d p r e s e r v a t i v e s (Rahn and Conn, 1944; Wolf and Westveer, 1950; Simon, 1952; Von Schelhorn, 1952; A l b e r t , 1957; B a n d e l i n , 1958; B e l l et a l . , 1959; de Navaree, 1959; E n t e r i k i n , 1961). 8 Since the d i s s o c i a t i o n constant of d i f f e r e n t p r e s e r v a t i v e s v a r i e s , t h e i r behavior i n d i f f e r e n t pH c o n d i t i o n s a l s o v a r i e s . Some are i n a c t i v a t e d by small increases i n pH w h i l e others are not in f l u e n c e d at a l l . When the pH of the environment i s below the pK , changes of pH are of l i t t l e consequence 3. but as the pH i s increased above the pK , higher concentrations are requ i r e d to produce a standard response (Simon, 1 9 5 2 ) . In the case of benzoic a c i d a constant c o n c e n t r a t i o n of u n d i s s o c i a t e d molecules does not produce the same response at d i f f e r e n t pH l e v e l s . This i n d i c a t e s that anions are a l s o s l i g h t l y t o x i c (Evans and Dunbar, 1 9 6 5 ; Anderson and Cho, 1 9 6 7 ) . ( 2 ) Oil-water p a r t i t i o n coefficient,;. P a r t i t i o n i n g has s i g n i f i c a n t e f f e c t on the a v a i l a b i l i t y of p r e s e r v a t i v e i n the aqueous phase (Husa and Radin, 1 9 3 2 ; A t k i n s , 1 9 5 0 ; A l l a w a l a and Riegelmann, 1 9 5 3 ; G a r r e t t and Woods, 1 9 5 3 ; Hibbott and Monks, 1 9 6 1 ; Bean, Heman-Ackah and Thomas, 1 9 6 5 ; and o t h e r s ) . Assuming that the amount i n the aqueous phase i s a c t i v e and i f some t r a n s f e r s to the o i l phase, s u f f i c i e n t a d d i t i o n a l p r e s e r v a t i v e should be provided to maintain the req u i r e d c o n c e n t r a t i o n i n water. Thus, the t o t a l p r e s e r v a t i v e to be added to a two-phase system can be c a l c u l a t e d by knowing the c o n c e n t r a t i o n r e q u i r e d i n the aqueous phase (C ) and knowing the volume ; 2 ° of each,phase ( V Q ; Q and Q ) and the d i s t r i b u t i o n c o e f f i c i e n t (Eq. 1 ) : t o t a l p r e s e r v a t i v e = CTT _ VTT _ + K° C„ _ V .., ^ 0 ^ 0 w ^ 0 o i l (Kostenbauder, 1962). ( 3 ) Oil-water r a t i o : The co n c e n t r a t i o n of p r e s e r v a t i v e i n the aqueous phase i s not c o n t r o l l e d by the p a r t i t i o n c o e f f i c i e n t alone, but by the i n t e r a c t i o n between the p a r t i t i o n c o e f f i c i e n t and the phase volume r a t i o (Bennett, 1 9 6 2 ; Bean and Heman-Ackah, 1 9 6 4 ; Bean, Heman-Ackah and Thomas, 9 1965; Bean, Konning and Malcolm, 1969). Table 2 shows the i n f l u e n c e of p a r t i t i o n c o e f f i c i e n t and phase volume r a t i o on the conce n t r a t i o n of preser -v a t i v e i n the aqueous and o i l phases of a two phase system. When the p a r t i t i o n c o e f f i c i e n t i s l e s s than one, the m a j o r i t y of the p r e s e r v a t i v e i s i n the aqueous phase and an increase i n the o i l - w a t e r r a t i o increases the aqueous phase co n c e n t r a t i o n . When the p a r t i t i o n c o e f f i c i e n t i s greater than one, most of the p r e s e r v a t i v e i s i n the o i l phase and an increase i n the o i l - w a t e r r a t i o reduces the con c e n t r a t i o n of p r e s e r v a t i v e i n the aqueous phase. When p a r t i t i o n c o e f f i c i e n t i s equal to one, changing the o i l - w a t e r r a t i o has no e f f e c t on the con c e n t r a t i o n of the p r e s e r v a t i v e i n e i t h e r phase. Thus, when s e l e c t i n g a compound f o r study as a p o s s i b l e p r e s e r v a t i v e f o r a product, both the p a r t i t i o n c o e f f i c i e n t and the o i l - w a t e r r a t i o must be considered. (4) I n t e r f a c i a l f a c t o r : Bean e_t a l . (1962, 1965) have shown that the b a c t e r i c i d a l a c t i v i t y of a given c o n c e n t r a t i o n of p r e s e r v a t i v e i n simple aqueous s o l u t i o n s i s l e s s than the b a c t e r i c i d a l a c t i v i t y of same concentra-t i o n of p r e s e r v a t i v e i n the aqueous phase of an o i l - w a t e r mixture. As the-r a t i o of o i l - w a t e r i n c r e a s e s , the b a c t e r i c i d a l a c t i v i t y of the p r e s e r v a t i v e i n c r e a s e s c o n s i d e r a b l y . I t was suggested that p r e s e r v a t i v e molecules are adsorbed at the o i l - w a t e r i n t e r f a c e , w i t h the p o l a r - p o r t i o n of the molecule p r o j e c t i n g i n t o the aqueous phase, and the nonpolar p o r t i o n p r o j e c t i n g i n t o the o i l phase. In t h i s way the conce n t r a t i o n of p r e s e r v a t i v e at the i n t e r -face i s higher than the bulk aqueous phase. When b a c t e r i a are added to such a system they are a l s o adsorbed at the o i l - w a t e r i n t e r f a c e , being heavier (diameter i n microns) than p r e s e r v a t i v e molecules (diameter i n X) they penetrate the aqueous phase more deeply than the p r e s e r v a t i v e molecules. I t Table 2 Influence of P a r t i t i o n C o e f f i c i e n t and Phase-Volume Ratio on Concentration of Pr e s e r v a t i v e i n Aqueous and O i l Phase of a Two-Phase System (Bean, Heman-Ackah and Thomas, 1965) K° w at 25C Oil/Water r a t i o 0.2 1.0 2.5 5.0 10.0 0.4% w/v phenol i n l i q u i d p a r a f f i n / w a t e r d i s p e r s i o n s 0.067 P r e s e r v a t i v e i n o i l % 0.031 0.050 0.080 0.080 0.176 P r e s e r v a t i v e i n water % 0.474 0.750 1.199 1.799 2.636 1% h y p o t h e t i c a l p r e s e r v a t i v e 1.000 P r e s e r v a t i v e i n o i l % 1.000 1.000 1.000 1.000 1.000 P r e s e r v a t i v e i n water % 1.000 1.000 1.000 1.000 1.000 4.0% w/v c h l o r o c r e s o l i n peanut o i l / w a t e r d i s p e r s i o n s 116.7 P r e s e r v a t i v e i n o i l % 22.96 P r e s e r v a t i v e i n water % 0.197 7.93 5.60 4.79 4.40 0.068 0.048 0.0411 0.038 11 i s t h e r e f o r e probable that p a r t of the b a c t e r i a l surface at the i n t e r f a c e i s i n contact w i t h a higher c o n c e n t r a t i o n of p r e s e r v a t i v e , but t h i s i s c e r t a i n l y not true f o r the w h o l e . c e l l . I f the b a c t e r i a were s t r o n g l y adsorbed at the i n t e r f a c e , as shown by Kamakaka (1956), then the preser-v a t i v e adsorbed at the i n t e r f a c e would have been much more e f f e c t i v e than was observed i n these s t u d i e s . (5) E f f e c t of temperature; Temperature i n f l u e n c e s the a c t i v i t y of p r e s e r v a t i v e s i n o i l - w a t e r d i s p e r s i o n s i n a complex manner (Bean and Heman-Ackah, 1965; Bean, Heman-Ackah and Thomas, 1965). Temperatures above 50° cause the death of v e g e t a t i v e c e l l s by p r o t e i h a c o a g u l a t i o n , enzyme i n a c t i v a t i o n , or both. In a d d i t i o n there are i n d i r e c t e f f e c t s of temperature on: ( i ) the o i l - w a t e r p a r t i t i o n c o e f f i c i e n t (Bean and Heman-Ackah, 1963); ( i i ) the o i l - w a t e r i n t e r f a c i a l a c t i v i t y which diminishes w i t h r i s e i n temperature (Heman-Ackah, 1965); ( i i i ) the v e l o c i t y of b a c t e r i c i d a l a c t i o n which increases w i t h temperature (Madsen and Nyman., 1907; Chick, 1908; Phelps, 1911). (6) E f f e c t of a d d i t i v e s : A d d i t i v e s such as propylene g l y c o l , g l y c e r i n , e t c . , are o f t e n included i n pharmaceutical and cosmetic emulsions as humectants. These a d d i t i v e s o f t e n b r i n g about an increased s o l u b i l i t y of p r e s e r v a t i v e i n the aqueous phase that reduces the o i l - w a t e r p a r t i t i o n c o e f f i c i e n t (Hibbott and Monks, 1961; Anderson and Cho, 1967). I t has been suggested (Hibbott and Monks, 1961) that t h i s makes more p r e s e r v a t i v e a v a i l a b l e i n the aqueous phase w i t h a consequent increase i n a n t i m i c r o b i a l a c t i v i t y . On the other hand, Anderson and Cho (1967) showed a r e d u c t i o n i n p r e s e r v a t i v e a c t i v i t y w i t h the a d d i t i o n of g l y c e r i n . They suggested that 12 although the i n c l u s i o n of g l y c e r i n reduces the o i l - w a t e r p a r t i t i o n c o e f f i c i e n t , i t a l s o reduces the a v a i l a b i l i t y of the p r e s e r v a t i v e to the m i c r o b i a l biophase. Consequently a higher c o n c e n t r a t i o n i s r e q u i r e d . In a d d i t i o n to reducing the o i l - w a t e r p a r t i t i o n c o e f f i c i e n t , Barr and T i c e (1957a,b) found that g l y c e r i n and s o r b i t o l supplement each other i n t h e i r i n h i b i t i o n of both b a c t e r i a and moulds. This e f f e c t was a t t r i b u t e d to an osmotic e f f e c t of the high concentrations of the humectants. Propylene g l y c o l , however, appeared to have a s p e c i f i c i n h i b i t o r y e f f e c t i n a d d i t i o n to i t s e f f e c t on the osmotic pressure of aqueous s o l u t i o n s . I t was concluded that propylene g l y c o l would have a s i g n i f i c a n t and u s e f u l p r e s e r v a t i v e e f f e c t , de Navarre (1962) reported that propylene g l y c o l was a r e l i a b l e p r e s e r v a t i v e at 16% v/v i n many cosmetic products because i t s a n t i m i c r o b i a l p r o p e r t i e s were three or four times that of the equivalent amount of g l y c e r i n . (b) Representation of d i s t r i b u t i o n data The d i s t r i b u t i o n of p r e s e r v a t i v e s between o i l - w a t e r systems can be.represented by the Nernst Equation (Nernst, 1891): K° = M (Eq.l) [Df] where K° i s the d i s t r i b u t i o n or p a r t i t i o n c o e f f i c i e n t ; [ b o ] , the concen-w t r a t i o n of p r e s e r v a t i v e i n the o i l phase; [ D f ] , the c o n c e n t r a t i o n of p r e s e r v a t i v e i n the aqueous phase. The r a t i o , K°, i s constant only f o r i d e a l s o l u t i o n s and i s most ' w' , c l o s e l y approximated when (a) the p r e s e r v a t i v e n e i t h e r d i s s o c i a t e s ( i o n i z e s ) nor a s s o c i a t e s i n e i t h e r phase, (b) the p r e s e r v a t i v e concentrations are approximately equal to a c t i v i t i e s , and (c) the two phases are completely immiscible (Reese et a l . , 1964). 13 When the p r e s e r v a t i v e i s monomeric i n the o i l phase, but i o n i z e s i n the aqueous phase, then the r a t i o of conc e n t r a t i o n of p r e s e r v a t i v e i n the o i l phase to the co n c e n t r a t i o n of unionized p r e s e r v a t i v e i n the aqueous phase w i l l be constant. O i l Water K dw •[HA]; [HA] (Eq.2) where K° i s the d i s t r i b u t i o n c o e f f i c i e n t f o r monomer; [HA] , i s the dw o' con c e n t r a t i o n of monomer i n the o i l phase; [HA]^, the co n c e n t r a t i o n of monomer, i n the aqueous phase. In case of a c i d p r e s e r v a t i v e s , such as benzoic a c i d and s o r b i c a c i d , the degree of i o n i z a t i o n i n the aqueous phase i s a f u n c t i o n of the pH of the aqueous phase and the i o n i z a t i o n constant, Ka, of the p r e s e r v a t i v e . This can be expressed [H +] [ A H Ka w [A~]. [HA] w Ka [HA] w w [H +] (Eq.3) (Eq.4) where [H ] i s the hydrogen : i o n c o n c e n t r a t i o n i n the aqueous phase. 14 The observed pH dependent d i s t r i b u t i o n c o e f f i c i e n t , K^, i s given by: K w [Do] [Df] [HA], [HAl + [ A - ] . w where [ A ~ ] w > i s the con c e n t r a t i o n of anion i n the aqueous phase. (Eq.5) S u b s t i t u t e [A ] from Eq.4 i n Eq.5 and rearrange w or K w [HA], 3A Ka [HA] [ H A V + + — ^ [ H + ] [HA], [HA] w 1 + Ka [H +] (Eq.6a) (Eq.6b) or tr w K dw 1 + Ka [H +] (Eq.6c) A p l o t of K° versus 1/(1 + Ka/[H +]) w i l l give a s t r a i g h t l i n e w i t h a slope w equal to K^ w, the monomer d i s t r i b u t i o n c o e f f i c i e n t . Rearranging Eq.6c i n double r e c i p r o c a l form gives 1 1 : + K w K dw Ka [H+] (Eq.7a) or Ka K w K dw K dw [ H + ] (Eq.7b) 15 A p l o t of 1/K° versus 1/[H +] gives a s t r a i g h t l i n e w i t h a slope of Ka/K° w and i n t e r c e p t equal to 1/K° w. Thus from Equations 6 and 7, the monomer d i s t r i b u t i o n c o e f f i c i e n t can be c a l c u l a t e d from the values of the observed pH dependent, c o n c e n t r a t i o n dependent d i s t r i b u t i o n c o e f f i c i e n t , K°, obtained over a range of hydrogen i o n c o n centrations. An equation s i m i l a r to Equations 6 and 7 has been derived by G a r r e t t and Woods (1953) to determine the monomer d i s t r i b u t i o n c o e f f i c i e n t when the p r e s e r v a t i v e i o n i z e s i n the aqueous phase. Ka + [H +] q K° + 1 Ka = dw_ ^ [ H + ] + * _ ( E q > 8 ) [Df] [D] [D] where q, i s the o i l - w a t e r r a t i o ; [D], the t o t a l c o n c e n t r a t i o n of p r e s e r v a t i v e i n the o i l - w a t e r system. Ka + [H +] + A p l o t of a gainst [H ] y i e l d s a s t r a i g h t l i n e w i t h a [Df] s l o p e , (q K j w + 1)/[D] and i n t e r c e p t , Ka/[D], The monomer d i s t r i b u t i o n c o e f f i c i e n t can thus be c a l c u l a t e d from slope and i n t e r c e p t over the range of hydrogen i o n c o n c e n t r a t i o n considered, as i l l u s t r a t e d below: (q K ° w + i ) m = :—: = slope (Eq.9a) [D] ' or q K ° w = m [D] - 1 (Eq.9b) or ,o m [D] - 1 Kdw " — - a ( E « ' 9 C ) Ka [D] 16 I n t e r c e p t . (Eq.lOa) or [D] = ^1 (Eq.lOb) c S u b s t i t u t i n g the value of [D] i n Eq.9c „o m Ka , /-T-. 11 \ K, = - 1 ( E q . l l a ) dw q c or K° = m K a - 1 C ( E q . l l b ) dw q c ^ Thus, from Eq.8, i f pH of the aqueous s o l u t i o n and the conce n t r a t i o n of p r e s e r v a t i v e i n the aqueous phase, [ D f ] , are known, the monomer d i s t r i b u t i o n c o e f f i c i e n t can be c a l c u l a t e d . U n l i k e Equations 6 and 7 there i s no need to c a l c u l a t e the observed pH dependent, concen-t r a t i o n dependent d i s t r i b u t i o n c o e f f i c i e n t . A l t e r n a t i v e l y , the monomer d i s t r i b u t i o n c o e f f i c i e n t , K^w» can be obtained according to Eq.2' by a n a l y s i s of the o i l phase and aqueous phase at a pH s u f f i c i e n t l y low to ensure that the p r e s e r v a t i v e e x i s t s completely i n the unionized form. When the p r e s e r v a t i v e i s monomeric i n the aqueous phase, but as s o c i a t e s to one species of m - mer (that i s , the a s s o c i a t i o n of more than one p r e s e r v a t i v e molecule) i n the o i l phase, three e q u i l i b r i u m constants are considered: 17 O i l Water K* [HA] om [HA] ,m (Eq.12) [HAl K dw [HA] w (Eq.2) K'' [HA] + m [HA] L o om w [HA] w (Eq.13) where K' , the pH independent, c o n c e n t r a t i o n dependent d i s t r i b u t i o n w c o e f f i c i e n t ; K', the a s s o c i a t i o n e q u i l i b r i u m constant f o r monomer and m - mer. Rearranging Eq.13 gives K w [HA] m [HA] L Jo L Jom [HA] [HA] L Jw w (Eq.14) or w m [HA] dw [HA] w (Eq.15) S u b s t i t u t i n g the value of [ H A ] o m from Eq.12 i n t o Eq.15 gives m K w m K' [HA] K° + 2 dw [HA] w (Eq.l6a) or 18 K'° = K° + m K' (K° ) m [ H A ] m _ 1 (Eq.l6b) w dw dw Jw I f [ H A ] w = [D'] = c o n c e n t r a t i o n of unionized p r e s e r v a t i v e i n aqueous phase. K'° = K° + m K ' ( K ° ) m [ D ; ] m _ 1 (Eq.17) w dw dw . f v n / The simplest case, i . e . , d i m e r i z a t i o n of p r e s e r v a t i v e i n o i l phase, corresponds to m=2 and then Eq.17 becomes K w = Kdw + 2 K ' ( K d w ^ ^ <E*-18> This l i n e a r r e l a t i o n s h i p between K'° and [D^] i s the one used by Gross and Schwarz (1930). I t s form has since been deduced by other authors ( S h i k a t a , 1931; P h i l b r i c k , 1934; Moelwyn-Hughes, 1940; Davies and Hallam, 1956). In the case of d i m e r i z a t i o n , a p l o t of K'° against w [D'] g i v e s a s t r a i g h t l i n e e x t r a p o l a t i n g to K° at [D'] = 0, and of slope 1 dw t 2 2K' (K, ) , from which the a s s o c i a t i o n constant, K', i s c a l c u l a t e d . In dw cases other than m = 2 Eq.17 leads to simple curves i f K'° i s p l o t t e d w against [D^.], showing m - m e r i z a t i o n of p r e s e r v a t i v e i n the o i l phase. 19 B. I n t e r a c t i o n of P r e s e r v a t i v e s w i t h Nonionic Surfactants This aspect has been the subject of numerous i n v e s t i g a t i o n s and has been reviewed elsewhere ( A l l a w a l a and Riegelmann, 1953; Wedderburn, 1964; Evans and Dunbar, 1965; Elworthy, Florence and Macfarlane, 1968). The f o l l o w i n g i s . a b r i e f account of the f a c t o r s p e r t i n e n t to an under-standing of t h i s problem. (a) P o t e n t i a t i o n of p r e s e r v a t i v e a c t i v i t y E a r l y observations of the i n a c t i v a t i o n and i n h i b i t i o n of germi-cides and p r e s e r v a t i v e s were made i n presence of i o n i c s u r f a c t a n t s ( F r o b i s h e r , 1927; Hampil, 1928; Ordal et a l . , 1941; Alexander and Tomlison, 1949; Bean and Berry, 1950, 1951). Most of these workers found that low concentrations of s u r f a c t a n t s ( i . e . , below c r i t i c a l m i c e l l e concentration) enhanced the e f f e c t s of germicides, w h i l e higher concentra-t i o n s l e d to v a r y i n g degrees of i n a c t i v a t i o n . Alexander and Trim (1946) st u d i e d the e f f e c t s of i o n i c s u r f a c t a n t s on the p e n e t r a t i o n of h e x y l r e s o r c i n o l i n t o the A s c a r i s worm. Maximum pe n e t r a t i o n was found to occur at the c r i t i c a l m i c e l l e concentration (CMC) which corresponds to the maximum concentration of monomolecularly dispersed s u r f a c t a n t . Schoog (1957) reported that the a c t i v i t y of hexachlorophene was increased by concentrations of a polyoxy--4 ethylene l a u r y l ether up to 10 M, but reduced by higher concentrations of the nonionic s u r f a c t a n t . Brown and Richards (1964) showed that the a n t i -b a c t e r i a l a c t i v i t y of c h l o r h e x i d i n e was enhanced by the presence of 0.02% polysorbate 80 but was reduced by 0.05% s o l u t i o n of the same s u r f a c t a n t . Bradshaw et a l . (1972) showed that low concentrations of Tween 80 po t e n t i a t e d the e f f e c t of c e t y l p y r i d i n i u m c h l o r i d e against E_. c o l i . 20 The exact mechanism of potentiation of preservative a c t i v i t y by surfactants, below t h e i r CMC, i s not well understood. I t has been ascribed to the surface-active properties of the surfactant monomers. The surfactant monomers possibly help i n the adsorption of preservative molecules at the bacteria/water i n t e r f a c e by reducing the i n t e r f a c i a l tension. (b) I n a c t i v a t i o n of preservative a c t i v i t y Since t h e i r introduction about 25 years ago, nonionic surfactants have found ever increasing use i n the preparation of s o l u b i l i z e d and emulsified systems. Despite t h e i r many advantages i n formulation, they have the serious disadvantage of suppressing, or even i n a c t i v a t i n g the e f f i c a c y of added preservatives. B o l l e and Mirimanoff (1950) were the f i r s t to point out the importance of t h i s phenomenon and showed that the antimicro-b i a l a c t i v i t y of methyl p-hydroxybenzoate was suppressed i n the presence of several s t r u c t u r a l l y d i f f e r e n t nonionic surfactants. They found that the surface-active sorbitan esters and polyoxyethylene sorbitan esters reduced the e f f e c t of methyl p-hydroxybenzoate, oxyquinoline s u l f a t e and dioxydichloro-diphenylmethane, whereassthe non surface-active Carbowax 1500, a polyethy-leneglycol polymer, had no i n a c t i v a t i n g e f f e c t . Lawrence and Erlandson (1953) and Erlandson and Lawrence (1953) found that Tweens and.certain other=nonionics reduced the germicidal e f f e c t of a number of phenolic compounds by a factor of 2000-5000 times. Barr and Tice (1957b) found that 5% Tween 20 rendered i n e f f e c t i v e a large number of phenolic substances when tested at the concentrations at which they are normally used. Similar i n a c t i v a t i o n s of commonly used preservatives i n the presence of a number of nonionic surfactants have been observed by de Navarre and Bailey (1956) and de Navarre (1957). The preservatives studied included benzoic a c i d , sorbic acid and methyl p-hydroxy-21 benzoate. Wedderburn (1958) assessed the e f f e c t of t h i r t y - s i x d i f f e r e n t nonionics on twenty-six a n t i m i c r o b i a l agents and found that although a l l the s u r f a c e - a c t i v e nonionics exerted some depressant e f f e c t , those which were not s u r f a c e - a c t i v e had n e g l i g i b l e e f f e c t s . The extent of the adverse e f f e c t was d i f f e r e n t f o r d i f f e r e n t combinations of nonionic s u r f a c t a n t and p r e s e r v a t i v e , and appeared to be r e l a t e d not only to the hy d r o p h i l e -l i p o p h i l e balance of the s u r f a c t a n t but a l s o to the chemical s t r u c t u r e of the p r e s e r v a t i v e . Blaug and Ahsan (1961b) examined the i n t e r a c t i o n of the methyl, e t h y l , p r o p y l and b u t y l p-hydroxybenzoates w i t h s e v e r a l nonionic macromolecules and reported that p-hydroxybenzoates of higher molecular weights were i n f l u e n c e d to a greater extent that those of lower molecular weight. In determining the bi n d i n g tendencies of the p-hydroxybenzoates w i t h Tween 80 (polyoxyethylene s o r b i t a n mono-oleate), Myrj 52 (polyoxyethy-lene monostearate), polyethylene g l y c o l 4000, 6000 and P l u r o n i c F-68 (polyethylene polypropylene g l y c o l ) , they confirmed that the h y d r o p h i l e -l i p o p h i l e balance s t r o n g l y i n f l u e n c e d the e f f e c t . Those compounds w i t h g r e a t e r l i p o p h i l i c tendencies reduced the e f f e c t i v e n e s s more than the more h y d r o p h i l i c macromolecules. Richards and Hardie (1972) showed that polysorbate 80 antagonized the a c t i v i t y of b i s (2-hydroxy-5 chloro-phenyl) s u l f i d e ( f e n t i c h l o r ) against exponential phase c u l t u r e s of S_. aureus, Pr . v u l g a r i s , and E. c o l i . The above s t u d i e s have demonstrated q u a l i t a t i v e l y the f a i l u r e of p r e s e r v a t i v e s i n the presence of nonionic s u r f a c t a n t s . I t i s evident that the i n a c t i v a t i o n r e s u l t s from an a s s o c i a t i o n of the p r e s e r v a t i v e molecule w i t h the macromolecule. Many q u a n t i t a t i v e s t u d i e s have been c a r r i e d out to determine the nature and extent of the i n t e r a c t i o n s which occur, from both 22 a p h y s i c a l and m i c r o b i o l o g i c a l point of view. In these systems an equilibrium may be postulated (Kostenbauder, 1962; Garrett, 1966) of the form of Eq.19, which for most of the i n t e r -actions reported has been shown to be r e v e r s i b l e . Preservative + Macromolecule " Preservative - Macromolecule (Eq.19) It i s generally agreed that the antimicrobial a c t i v i t y of such systems depends mainly on the concentration of unbound or free preser-vative (Wedderburn, 1964; M i t c h e l l , 1964) rather than the t o t a l concen-t r a t i o n present. However, Humphreys, Richardson and Rhodes (1968) showed that the a n t i m i c r o b i a l a c t i v i t y of a concentration of preservative i n aqueous so l u t i o n was l e s s than the same concentration of preservative i n an aqueous phase of a surfactant s o l u t i o n . They concluded that a n t i m i c r o b i a l a c t i v i t y i s not only a function of the concentration of preservative i n the aqueous phase but some a d d i t i o n a l factors also control the extent of a n t i m i c r o b i a l a c t i v i t y . (c) Mechanism of i n a c t i v a t i o n of preservatives by nonionic surfactants An examination of the l i t e r a t u r e reveals that the mechanism of i n t e r a c t i o n has been a subject of much debate, and the controversy has centred between two schools of thought. One maintains that the i n t e r -action between preservative and surfactant i s due to complex formation, while the other believes that i n t e r a c t i o n i s due to p a r t i t i o n i n g of preservative into surfactant m i c e l l e s . Higuchi and Lach (1954) reported the formation of hydrogen bonded complexes between polyethylene g l y c o l s and phenols and between 23 polyethylene g l y c o l s and organic acids. Since most nonionic surfactants have polyethylene g l y c o l chains, many authors (Guttman and Higuchi, 1956; Mulley and Metcalf, 1956; de Navarre, 1956, 1957; Barr and Tice, 1957b) have a t t r i b u t e d both the solvent properties and the i n a c t i v a t i o n of preservative to complex formation. Evans (1964) showed that complex formation between surfactant monomer and preservative i s u n l i k e l y and suggested that i n a c t i v a t i o n a r i s e s from s o l u b i l i z a t i o n of preservative within the surfactant m i c e l l e s . Mulley (1964) c o l l e c t e d evidence from a number of sources which indicates that the s o l u b i l i z a t i o n of a wide range of solutes i n nonionic surfactants can be treated as a s o l u t i o n process within the hydrocarbon-like i n t e r i o r of the m i c e l l e . He considered that the data does not support suggestions that s o l u b i l i z a t i o n i s c o n t r o l l e d by more s p e c i f i c factors such as complex formation. Some workers (Evans and Dunbar, 1965; Wedderburn, 1964) suggested that since i n a c t i v a t i o n occurs with preservatives and surfactants offisuch diverse chemical structures, s o l u b i -l i z a t i o n rather than s p e c i f i c complexing i s a more probable explanation. Support for these authorssis found i n the f a c t that non-surface active macromolecules i n t e r a c t with preservatives to a much l e s s e r extent than surfactant molecules. Kostenbauder (1962) maintains that i t i s unnecessary to d i s t i n g u i s h between the mechanism of complex formation and m i c e l l a r s o l u b i l i z a t i o n and considers that s o l u b i l i z a t i o n and m i c e l l e formation i t s e l f f a l l within the broad scope of complex formation described by Higuchi and Lach (1954), because both processes obey the law of mass action. M i c e l l e s of nonionic surfactants appear to provide i d e a l conditions for a s s o c i a t i o n with preservatives. They a f f o r d the p o s s i b i l i t y for hydrogen bonding and s o l u b i l i z a t i o n within the hydrocarbon-like i n t e r i o r of the m i c e l l e . I t seems l i k e l y that under su i t a b l e conditions both these mechanisms may operate simultaneously. 24 (d) P o s s i b l e s i t e s f o r the i n t e r a c t i o n of p r e s e r v a t i v e s i n a s u r f a c t a n t m i c e l l e The s i t e of i n c o r p o r a t i o n of s o l u b i l i z a t e i n a s u r f a c t a n t m i c e l l e i s b e l i e v e d to be c l o s e l y r e l a t e d to i t s chemical nature, as w e l l as the chemical nature of the s u r f a c t a n t . In aqueous s o l u t i o n i t i s g e n e r a l l y accepted that nonpolar s o l u b i l i z a t e s , e.g., a l i p h a t i c hydro-carbons, are d i s s o l v e d i n the hydrocarbon core of the m i c e l l e ( F i g . 1 ( a ) ) . Semipolar or p o l a r s o l u b i l i z a t e s , e.g., f a t t y a c i d s and a l k a n o l s , are taken up i n what i s o f t e n termed the p a l i s a d e (or oxyethylene) l a y e r of the m i c e l l e , , o r i e n t e d w i t h t h e i r hydrophobic moie t i e s towards the centre of the m i c e l l e and t h e i r p o l a r groups i n i t s surface. Riegelmanv et a l . (1958) studied v a r i o u s aromatic compounds i s o l u b i l i z e d i n aqueous s o l u t i o n s of potassium l a u r a t e , dodecyl amine hyd r o c h l o r i d e and a polyoxyethylene ether of l a u r y l a l c o h o l using UV spectro-scopy and concluded that ethyl-benzene was incorporated i n the hydrocarbon p o r t i o n of the m i c e l l e ( a ) , o S n i t r o a n i l i n e was l o c a t e d at a p o s i t i o n of short p e n e t r a t i o n i n t o the p a l i s a d e l a y e r (b), azobenzene, naphthalene and anthracene were i n a p o s i t i o n of deep p e n e t r a t i o n of p a l i s a d e l a y e r ( c ) , and dimethyl phthalate was adsorbed on the surface of the m i c e l l e (d), ( F i g . 1).. Mulley and Metcalf (1956) suggested from t h e i r UV spectroscopic s t u d i e s that c h l o r o x y l e n o l s o l u b i l i z e d i n the m i c e l l e s of cetomacrogol formed hydrogen bonds between phenolic hydroxyl groups and oxygen atoms of the polyoxyethylene chains ( F i g , 1 ( e ) ) . F i g . 1. P o s s i b l e s i t e s of i n c o r p o r a t i o n of s o l u b i l i z a t e i n a m i c e l l e (Elworthy e t _ a l . , 1958): (a) i n the hydrocarbon core; (b) short p e n e t r a t i o n of the p a l i s a d e l a y e r ; (c) deep pe n e t r a t i o n of the p a l i s a d e l a y e r ; (d) ads o r p t i o n on the surface of the m i c e l l e ; (e) i n the polyoxyethylene s h e l l of the m i c e l l e of a nonionic' s u r f a c t a n t ; ( f ) at the j u n c t i o n of hydrocarbon core and polyoxyethylene chain of nonionic s u r f a c t a n t . A, m i c e l l e of i o n i c s u r f a c t a n t . B, m i c e l l e of nonionic s u r f a c -t a n t . N 3 Ul 26 Anderson and Slade (1965) compared the UV spectra of benzoic acid in hexane, water and Myrj 59 solutions. In hexane a typical benzehoid pattern was obtained, whereas the spectra in a l l surfactant solutions were similar to that in water and showed no fine structure? From these observations the authors concluded that solubilized benzoic acid was located largely in a polar environment, i.e., the oxyethylene layer of the micelle (Fig. 1(e)). Donbrow and Rhodes (1966) used UV and NMR spectroscopy to examine the location of benzoic acid in cetomacrogol micelle. They favoured the view that the position of the benzoic acid molecules in the micelle was at the junction of the hydrocarbon core and the polyoxyethylene chains (Fig. 1(f)), with the benzene ring enclosed in the former and the carboxylic acid group protruding outwards. Such a position would s t i l l allow for hydrogen bonding between carboxyl group and the innermost ether oxygen. Their previous potentiometric studies also supported the same view (Donbrow and Rhodes, 1964). In another study, Donbrow et a l . (1967) employed potentio-metric and NMR techniques which suggested that m- and p-hydroxybenzoic acids were located at the polyoxyethylene chains of cetomacrogol micelle (Fig. 1(e)). Jacobs et a l . (1971), examining the interaction of phenol with cetomacrogol using NMR spectroscopy, suggested that phenol was located in the polyoxyethylene region of the cetomacrogol micelle. Corby and Elworthy (1971a,b) identified the sites of s o l u b i l i -zation of esters of p-hydroxybenzoic acid in a cetomacrogol micelle using UV, NMR, solubility and viscometric techniques. The sites of solubilization were suggested to be as follows: p-hydroxybenzoic acid was wholly 27 s o l u b i l i z e d deep i n the oxyethylene layer of the m i c e l l e , ethyl p-hydroxy-benzoate -was s o l u b i l i z e d mostly i n the oxyethylene layer situated adjacent to the core, with some s o l u b i l i z a t i o n occurring within the core, b u t y l p-hydroxybenzoate was s o l u b i l i z e d mainly at the oxyethylene layer with the bu t y l chain i n the core while some s o l u b i l i z a t e was wholly present i n the core, methyl p-methoxybenzoate was mostly dissolved i n the m i c e l l a r core, but some was also present i n the oxyethylene layer, benzene was s o l u b i l i z e d i n a s i m i l a r manner to methyl p-methoxybenzoate. (e) E f f e c t of preservatives on the m i c e l l a r molecular weight of surfactants The e f f e c t of s o l u b i l i z a t e on m i c e l l a r s i z e has been examined i n r e l a t i v e l y few systems. In a l i g h t - s c a t t e r i n g study of s o l u b i l i z a t i o n by hexadecyltrimethylammonium bromide (Hyde and Robb, 1964) i t was shown that the incorporation of increasing amounts of the non-polar molecules, decane, octane and cyclohexane, caused a pronounced increase i n the m i c e l l a r molecular weight (Mu). This was due to an increase i n the number of s o l u b i l i z a t e and surfactant molecules i n each m i c e l l e . However, the s o l u b i l i z a t i o n of the polar molecule, octanol, although increasing the Mu, caused a decrease i n the number of surfactant molecules i n each m i c e l l e . Nakagawa et a l , (1959, 1960) found that the s o l u b i l i z a t i o n of decane and decanol by three methoxypolyoxy.ethy.lene decyl ethers resulted i n increases i n the m i c e l l a r weight of the micelles of these nonionic surfactants. Each weight increase was a consequence of increases i n the amount of s o l u b i l i z a t e and surfactant per m i c e l l e . V i s c o s i t y and sedimentation studies of the s o l u b i l i z a t i o n of 1,2,4-trichlorobenzene and tolu rene by cetylpyridinium c h l o r i d e (Smith and Alexander, 1957) have indicated an increase i n Mu and i n 28 m i c e l l a r asymmetry with- increase i n s o l u b i l i z a t e concentration up to a maximum, a f t e r which further s o l u b i l i z a t e promoted the formation of a more spheri c a l m i c e l l e which existed i n equilibrium with the r o d - l i k e micelles produced i n i t i a l l y . In contrast, the s o l u b i l i z a t i o n of methylcyclohexane by the same surfactant resulted i n only a small regular increase i n Mu and v i s c o s i t y . In most cases, the differences i n the e f f e c t s of the various s o l u b i l i z a t e s on the m i c e l l e s i z e and shape have been a t t r i b u t e d to d i f f e r e n c e s i n the l o c a t i o n of s o l u b i l i z a t e within the m i c e l l e . Recently, Attwood, Elworthy and Kayne (1971) examined the s o l u b i l i z a t i o n of decane, eth y l p-hydroxybenzoate, methyl anisate and p-hydrox.ybenzoic•acid by aqueous m i c e l l a r solutions of cetomacrogol using membrane osmometry and v i s c o s i t y techniques. The e f f e c t of s o l u b i l i z a t e s on the number average m i c e l l a r molecular weight, Mn, was r e l a t e d to t h e i r s i t e of incorporation i n the m i c e l l e (Fig. 2). Decane and methyl anisate were s o l u b i l i z e d i n the hydro-carbon core of the micelles and both compounds produced an increase i n Mn up to a maximum value of 2.0 x 10^, at a s o l u b i l i z a t e concentration of approximately 80% of the saturation l i m i t f or each compound. This increase was shown to r e s u l t from an increase i n the number of molecules of both s o l u b i l i z a t e and of surfactant per m i c e l l e . Further a d d i t i o n of s o l u b i l i -zate, to produce a saturation l e v e l i n excess of 80%, r e s u l t s i n a decrease i n m i c e l l a r s i z e i n both systems. The s o l u b i l i z a t i o n of e t h y l p-hydroxybenzoate and p-hydroxybenzoic acid was thought to involve the oxyethylene region of the m i c e l l e and both the s o l u b i l i z a t e s cause an increase i n Mn owing to the i n c l u s i o n of s o l u b i l i z a t e into the m i c e l l e , the number of molecules of surfactant per m i c e l l e being unaffected by the s o l u b i l i z a t i o n process. 29 2.2 IT) I o 30 40 50 60 70 Saturation (%) 80 90 100 Figure 2. Variation of micellar molecular weight, Mn, with the degree of saturation of cetomacrogol micelle witho, decane; Ar , methyl anisate; f , p-hydroxybenzoic acid and&, ethyl p-hydroxybenzoate (Attwood, Elworthy and Kayne, 1971). 30 (f) Representation of interaction data The mathematical treatment of results for the interaction between preservatives and surfactants has been presented in a variety of ways. Since the mechanism of interaction proposed governs the mathematical model used in the calculation, literature on the subject has been contro-versial. A more detailed review and evaluation of these approaches is given elsewhere (Kazmi and Mitchell, 1971a). The interaction between preservative and surfactant can be represented by the law of mass action. Consider a simple situation where a macromolecule CM) combines with one molecule of preservative (D) to form a complex (DM), then at equilibrium K D + M *" , DM (Eq.19) the association constant, K, is defined by the following equation: [Db] K = (Eq.20a) [Mf][Df0 or [Db] = K [Df][Mf] (Eq.20b) where [Df], the concentration of free preservative; [Db], the concentration of preservative bound with macromolecule; [Mf], the concentration of free macomolecule. But [M] = [Mf] + [Db] (Eq.21a) or Total concentration _ Free concentration + Bound concentration of macromolecule of macromolecule of macromolecule or [Mf] = [M] - [Db] (Eq.21b) S u b s t i t u t e the value of [Mf] i n Eq.20b and rearrange [Db] = K [Df] | [M] - [Db] j (Eq.22) [Db] K [Df] [M] 1 + K :[Df ] (Eq.23) I f the macromolecule has n independent b i n d i n g s i t e s and each s i t e has the same i n t r i n s i c a f f i n i t y f o r p r e s e r v a t i v e and i s not i n f l u e n c e d by i t s neighbours, the Eq.23 i s m u l t i p l i e d by ! n ' , and [Db] n K [Df] r = == (Eq. 24) [M] 1 + K [Df] where r represents molar r a t i o , i . e . , the number of moles of p r e s e r v a t i v e bound per mole of macromolecule. This r a t i o i n d i c a t e s the extent of b i n d i n g . I f a l l the monomers i n a s u r f a c t a n t m i c e l l e behave independently and the i n t e r a c t i o n of p r e s e r v a t i v e w i t h the m i c e l l e does not change the m i c e l l a r molecular weight, then.Eq.24 can be u t i l i z e d to represent the i n t e r -a c t i o n of p r e s e r v a t i v e w i t h s u r f a c t a n t m i c e l l e s . In t h i s case, r w i l l give number of moles of p r e s e r v a t i v e bound [D^], per mole of m i c e l l e [M], n, the number of b i n d i n g s i t e s per m i c e l l e , and K, the a s s o c i a t i o n constant f o r i n t e r a c t i o n w i t h the m i c e l l e . Assume, as a f u r t h e r extension, that two e n t i r e l y d i f f e r e n t c l a s s e s of s i t e s e x i s t i n the s u r f a c t a n t molecule and that as before, a l l s i t e s are independent and s i t e s w i t h i n the c l a s s are e q u i v a l e n t . A s i m i l a r treatment can be used to d e r i v e an expression f o r ' r ' f o r t h i s case. Thus, 32 r . J l A^ f l + ( E q . 2 5 ) 1 + ^ [ D ^ ] 1 + K 2[D f ] where: ti ^ and = number of independent binding s i t e s of class I and class I I , respectively, on the surfactant molecule, and = i n t r i n s i c association constant for the binding of a molecule of preservative to one of the binding sit e s of class I and class I I , respectively, i n the surfactant molecule. In t h i s work, Eq.25 w i l l be used to characterize interaction of various preservatives with some nonionic surfactants. A general expression which describes binding to 'm' different classes of site s i s : r = ) 1 1 (Eq.26) i - i 1 + W The t o t a l preservative concentration, [D ], required i n a surfactant solution can be calculated by substituting the values of binding parameters (n's and K's), [M] and [D^] into Eq.27, i f only one class of binding sit e s i s involved i n the interaction, or Eq.28, i f two classes of binding s i t e s are assumed to be involved i n the interaction. T n K [ M ] 1 [D.] = [D ] { 1 + > (Eq.27) t f 1 1 + K [D f] J j n K [M] ,n K [M ] 1 [ D j = [ D j { 1 + " — i - i + ^ - k \ ( E q . 2 8 ) t f [ 1 + K l [ D f ] 1 + K 2[D f] J 33 C. I n t e r a c t i o n of P r e s e r v a t i v e Mixtures w i t h Nonionic Surfactants In recent years much emphasis has been given to the use of a combination of p r e s e r v a t i v e s r a t h e r than a s i n g l e p r e s e r v a t i v e f o r the p r e s e r v a t i o n of cosmetic and pharmaceutical p r e p a r a t i o n s . (Boehm e t a l . , 1957, 1959, 1968, 1970; Gershenfeld, 1963; G a r r e t t , 1966; Casely et a l . , 1968; Kostenbauder, 1968; Richards, 1971; Richards and Hardie, 1972; Richards and MacBride, 1971, 1973; Boehm and Maddox, 1971; Parker, 1971, 1972, 1973; P r o s e r p i o , 1972; Fontana and P r o s e r p i o , 1972). The r a t i o n a l e s f o r combinations are that the spectrum of a c t i v i t y can be increased; that : the p h y s i o l o g i c a l l y harmful e f f e c t s of a dose of one p r e s e r v a t i v e alone g i v i n g an equivalent e f f e c t may be averted; that the development or m o d i f i -c a t i o n of the r e s i s t a n c e of an organism to one p r e s e r v a t i v e alone may be prevented; that response may exceed p r e d i c t i o n from the separate p r e s e r v a t i v e a c t i o n or from any concentrations of one p r e s e r v a t i v e alone; that convenience of a d m i n i s t r a t i o n of smaller amounts or economic savings may r e s u l t ( G a r r e t t , 1966). The i n t e r a c t i o n of p r e s e r v a t i v e s w i t h nonionic s u r f a c t a n t s has been s t u d i e d e x t e n s i v e l y ( P a t e l and Kostenbauder, 1958; M i t c h e l l and Brown, 1966; Kazmi and M i t c h e l l , 1971a, 1973, and many more). L i t t l e i n f o r m a t i o n i s a v a i l a b l e concerning the s o l u b i l i z a t i o n of p r e s e r v a t i v e mixtures by nonionic s u r f a c t a n t s . Recently, Crook and Brown (1973) reported the i n t e r -a c t i o n of s e v e r a l p r e s e r v a t i v e combinations w i t h cetomacrogol using a s o l u b i l i t y technique. They found that the s o l u b i l i t y of a given p r e s e r v a t i v e i n cetomacrogol s o l u t i o n s was a l t e r e d by the a d d i t i o n of another p r e s e r v a t i v e . Since p r e s e r v a t i v e mixtures are o f t e n employed i n cosmetic and . 34 pharmaceutical preparations containing nonionic surfactants, the present study was undertaken to examine the binding behavior of preservative mixtures with a nonionic surfactant, cetomacrogol. The binding was studied in less than saturated conditions because the effective concen-tration of most preservatives in the aqueous phase of a surfactant solution l i e s below the saturation solubility in water. D. Theory of Competitive Binding of Drugs with Macromolecules The competitive binding of drugs with proteins (Meyer and Guttman, 1968) and enzymes (Brand e_t a l . , 1967) occurs when two or more drugs compete for the same locus on the macromolecule. Mathematical methods used to express the displacement of one drug by another drug from protein binding sites are well established (Klotz e_t a l . , 1948; Karush, 1950; Cogin and Davis, 1951; Meyer and Guttman, 1970). In this investi-gation, an attempt has been made to test these mathematical methods for the binding of preservative mixtures with the nonionic surfactant cetomacro-gol. A f i t of the binding data to these mathematical models w i l l enable a prediction to be made of the total concentration of preservative mixture required in a surfactant solution to provide the desired concentration of each preservative free in the aqueous phase. The interaction of a preservative (D) with surfactant (M) can be characterized by Eq.25, which can be rewritten in the form of Eq.29: n K [D ] n K [D ] r = - + L _ (Eq.29) 1 + K d l [D f] 1 + K d 2 [D f] In the presence of another preservative (C), the competitor, Eq.29, can be 35 written: n l K d l . n2 Kd2 t° f ] r = + ^ ± —, (Eq.30) 1 + K d l [D f] + K c l [C f] 1 + K d 2 [D f] + K c 2 [C f] where [C^] i s the concentration of free competitor, and are the intrinsic association constants for the binding of a molecule, of competitor to one of the binding sites of class I and class I I , respectively, in the surfactant molecule. If n^ and are equivalent and independent, then: n l K d l 3 n2 Kd2 D f ] rd " ' + (Eq.31) I + K d i:[D f] 1 +K d 2 [D f] where and are given by Eq.s 32a and 32b respectively: K i - = (Eq.32a) d l 1 + K c i ' [ C f ] Kd2 = (Eq.32b) 1 + K C 2 [C f] Equation 31 implies that in the presence of a constant concentra-tion of C, the binding of D to M follows the same pattern as that indicated in Eq.29, except that and K d 2 values w i l l be lower than that observed for K,.. and K J O, and are a function of C. If K,t and K,„ are known, and a l a l dV K' and K' are-determined for a known value of C, then K ' and K 0 0 can be dl az L i t.z calculated from Eq.s 33a and 33b respectively: 1 / KH1 K r l = : — - 1 | (Eq.33a) tC f] \ K d l 36 1 K "d2 K, C2 - 1 (Eq.33b) ^ \ K d d2 The above equations are derived i n terms of r ^ , but analogous equations can be derived i n terms of r , the moles of species C bound KJ per mole of surfactant. E. Interaction of a Preservative with Mixtures of Nonionic Surfactants The use of more than one nonionic surfactant i s common i n s o l u b i l i z e d and emulsified systems, Surfactant mixtures o f f e r many advantages over i n d i v i d u a l surfactants, e.g., adjustment to a desired HLB value ( G r i f f i n , 1949), improvement of emulsion s t a b i l i t y !(Boyed, Parkinson and Sherman, 1972), and attainment of desired r h e o l o g i c a l properties (Barry, 1969; Barry and Saunders, 1972; Eccleston, Barry and Davis, 1973). While some studies have been made of the s o l u b i l i z a t i o n behavior of mixtures of anionic and nonionic surfactants (Fukuda and Taniyama, 1958; Saito and H i r a t a , 1959; Narasaki, 1961; Narasaki and Suzuki, 1962; H a l l and Soudah, 1966), l i t t l e information i s a v a i l a b l e regarding the i n t e r a c t i o n of solutes with mixtures of nonionic surfactants. H a l l (1963) studied the e f f e c t of polysorbate 20 on the s o l u b i l i z i n g power of polysorbate 80 and demonstrated that polysorbate 20 decreased the " c r i t i c a l m i s c i b i l i t y r a t i o " of s a l i c y l i c acid i n a l i n e a r fashion. c e r t a i n nonionic surfactants and t h e i r mixtures i s reported. Binding para-meters which characterize the i n t e r a c t i o n of a preservative with each i n d i v i d u a l surfactant have been used to predict the binding behavior of surfactant mixtures. In the present i n v e s t i g a t i o n , the i n t e r a c t i o n of c h l o r o c r e s o l with 37 F. Representation of Interaction Data for the Binding of a Preservative with Mixtures of Nonionic Surfactants For the i n t e r a c t i o n of a preservative with a surfactant, [D t] f o r a given [D^] can be calculated using Eq.27. If there are several types of surfactants of concentrations M^, M„, M 0 ... M with number of binding s i t e s per molecule n., n„, n 0 ... n 2 3 m 1 2 3 m and with i n t r i n s i c a s s o c i a t i o n constants K, , K~, K„ .... K , i t follows by 1 2 3 m reasoning s i m i l a r to that for Eq.27 that: [ n K [M ] n K [M ] n K [ M j "I [D ] = [D ]< 1 + 1 1 1 + £ + . . . + J 2 J 5 2 \ t L 1 + \ [D f] 1 + K 2 [D f] 1 + K m [D f] J or f » n K [ M ] I I i = l 1 + K i t D f ] J (Eq.34) m , K , Lw,j | (Eq.35) S i m i l a r l y for a mixture of surfactants, each with two classes of binding s i t e s : f n l i K l i n 2 i K 2 i [ M i ] 1 [ D J = [D ]< 1 + ) - ^ - ^ ^ _ + ; 2 1 2 1 1 ) L ± . V i l' + K i i C D f ] l + K 2 i [ D f ] J. (Eq.36) For a mixtureoof two types of surfactants, the Eq.36 can be written: n l K l E M I ] ^ . [ M ^ . njKj [M^] . f n K. K, 2 [D f] 1 + K 2 • [ ° f ] 1 + K i [ ° f 3 n 2 K 2 [ M I I ] 1 1 + KJ [D f] J (Eq.37) 38 where: Mj. = conc e n t r a t i o n of s u r f a c t a n t of type I . = co n c e n t r a t i o n of s u r f a c t a n t of type I I . n^ and n| = number of independent b i n d i n g s i t e s of c l a s s I on the s u r f a c t a n t molecule of type I and I I r e s p e c t i v e l y . n 2 and n^ = number of independent b i n d i n g s i t e s of c l a s s I I i n the s u r f a c t a n t molecule of type I and I I r e s p e c t i v e l y , and = i n t r i n s i c a s s o c i a t i o n constants f o r the bi n d i n g of a molecule of p r e s e r v a t i v e to one of the bi n d i n g s i t e s of c l a s s I i n s u r f a c t a n t molecules of type I and I I r e s p e c t i v e l y . K 2 and K 2 = i n t r i n s i c a s s o c i a t i o n constants f o r the b i n d i n g of a molecule of p r e s e r v a t i v e to one of the bi n d i n g s i t e s of c l a s s I I i n s u r f a c t a n t molecules of type I and I I r e s p e c t i v e l y . In the present work, Eq.37 has been used to c h a r a c t e r i z e the i n t e r a c t i o n of a p r e s e r v a t i v e w i t h a mixture of two types of s u r f a c t a n t s . The values of b i n d i n g parameters (n's and K's) f o r the s u b s t i t u t i o n of Eq.37 were estimated using Eq.25, which describes the b i n d i n g of a p r e s e r v a t i v e w i t h a s u r f a c t a n t , assuming that•two c l a s s e s of bi n d i n g s i t e s are i n v o l v e d i i n the i n t e r a c t i o n . G. D i s t r i b u t i o n and A n t i m i c r o b i a l A c t i v i t y of P r e s e r v a t i v e s i n E m u l s i f i e d Systems (a) I n t r o d u c t i o n Factors a f f e c t i n g the a n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s i n e m u l s i f i e d systems are much more complex than simple aqueous systems. Some notable reviews on the subject have appeared i n the l i t e r a t u r e (Tice and 39 Barr, 1958; Kostenbauder, 1962; Bennett, 1962; de Navarre, 1962; Wedderburn, 1964). Aoki et a l . (1957) studied various factors important i n the preservation of hydrophilic ointment (U.S.P. XV; J.P. VI) and si m i l a r emulsions by p-hydroxybenzoates. They found that the amount of ester required was dependent not only on the nature of the ester but also on the nature of components i n the o i l and water phases. Matsumoto and Aoki (1962) l a t e r found that the degree of i n a c t i v a t i o n of p-hydroxybenzoates was dependent upon the r e l a t i o n s h i p between the properties of .the ester, the surfactant and the o i l y substance to be incorporated i n the emulsion. O i l y substances, such as isopropyl myristate, o l i v e o i l , l a u r y l a l c o h o l , xylene and d i b u t y l phthalate, considerably a l t e r e d the a c t i v i t y of the esters. Propyl p-hydroxybenzoate was found to be more subject to i n a c t i v a -t i o n than the methyl ester. I t was suggested; that the propyl p-hydroxy-benzoate was inacti v a t e d by ready s o l u b i l i z a t i o n into the l i p o p h i l i c part of the m i c e l l e . Kostenbauder (1962) derived an equation which involved the use of the oil-water p a r t i t i o n c o e f f i c i e n t and preservative-macromolecule binding data (see Eq.44). The equation permitted a c a l c u l a t i o n to be made of the amount of preservative required i n an emulsion to provide a concen-i t r a t i o n of preservative i n the aqueous phase s u f f i c i e n t to i n h i b i t microbial growth. Garrett (1966) subsequently developed a more comprehensive mathe-matical model, which quantified a l l the pertinent factors responsible for the i n a c t i v a t i o n of preservatives i n heterogeneous systems. Anderson and Cho (1967) reported on the distribution.and a c t i v i t y of benzoic a c i d in,oil-water systems emulsified with 0.1% polyoxyethylene l a u r y l ether ( B r i j 35). The antifungal a c t i v i t y of benzoic acid was re l a t e d to i t s concentration i n the aqueous phase. Bean, Konning and Malcolm (1969) found close agreement between 40 p r e d i c t i o n s made using an equation s i m i l a r to that of Kostenbauder (1962) and the r e s u l t s obtained from m i c r o b i o l o g i c a l s t u d i e s . They concluded that the a c t i v i t y of p r e s e r v a t i v e s i n e m u l s i f i e d systems was r e l a t e d to the c o n c e n t r a t i o n f r e e i n the aqueous phase. P a t e l and Romanowski (1970) a l s o v e r i f i e d Kostenbauder's equation (1962) by an i n - v i t r o m i c r o b i o l o g i c a l procedure. They showed that the f u n g i s t a t i c a c t i v i t y of methyl and p r o p y l p-hydroxybenzoates i n e m u l s i f i e d systems was p r i m a r i l y a f u n c t i o n of the fr e e paraben co n c e n t r a t i o n i n the aqueous phase. M a r s z a l l (1972) reviewed v a r i o u s mathematical models used f o r p r e d i c t i n g the re q u i r e d p r e s e r v a t i v e c o n c e n t r a t i o n i n e m u l s i f i e d systems. (b) C a l c u l a t i o n of t o t a l p r e s e r v a t i v e c o n c e n t r a t i o n r e q u i r e d i n an emulsion When a p r e s e r v a t i v e i s added to a simple emulsion c o n s i s t i n g of o i l , water and s u r f a c t a n t phases, p a r t of i t i s p a r t i t i o n e d i n t o the o i l phase and p a r t i s complexed or s o l u b i l i z e d w i t h i n the s u r f a c t a n t m i c e l l e s . The r e s t . o f the p r e s e r v a t i v e remains i n the aqueous phase. P r e s e r v a t i v e p a r t i t i o n e d . - i n t o the o i l phase, or bound to the s u r f a c t a n t m i c e l l e s , has l i t t l e a n t i m i c r o b i a l a c t i v i t y and the a n t i m i c r o b i a l a c t i v i t y i s s a i d to depend mainly on the co n c e n t r a t i o n of p r e s e r v a t i v e i n the aqueous phase (Anderson and Cho, 1967; Bean, Konning and Malcolm, 1969; P a t e l and Romanowski, 1970). Thus from the physico-chemical parameters governing the d i s t r i -b u t i o n of p r e s e r v a t i v e s i n o i l - w a t e r systems and the bi n d i n g of p r e s e r v a t i v e w i t h s u r f a c t a n t m i c e l l e s i t should be p o s s i b l e to c a l c u l a t e the t o t a l c o n c e n t r a t i o n of p r e s e r v a t i v e r e q u i r e d i n an emulsion to provide a concen-t r a t i o n of p r e s e r v a t i v e i n the aqueous phase s u f f i c i e n t to i n h i b i t m i c r o b i a l growth. The t o t a l amount of p r e s e r v a t i v e i n an emulsion, W, i s given by W = [Dt] Vw + [Do] Vo (Eq.38) where [Dt] i s the t o t a l c o n c e n t r a t i o n of p r e s e r v a t i v e i n aqueous phase, [Do] i s the conc e n t r a t i o n of p r e s e r v a t i v e i n o i l phase, Vw i s the volume of the aqueous phase and Vo i s the volume of the o i l phase. But W = [D] (Vo + Vw) and [Dt] = [Df] + [Db] where [D] i s the t o t a l c o n c e n t r a t i o n of p r e s e r v a t i v e i n the emulsion, [Df] i s the con c e n t r a t i o n of f r e e p r e s e r v a t i v e i n the aqueous phase and [Db] i s the conc e n t r a t i o n of bound p r e s e r v a t i v e i n the aqueous phase. S u b s t i t u t e the values of W and [Dt] i n Eq.38 and rearrange [D] (Vo + Vw) = [Df] + [Db] j Vw + [Do] Vo (Eq.39) [D] = |^[Df] + [Db] J Vw + [Do] Vo^|/(Vo + Vw) (Eq.40) But [Db] = n K [M] [Df] / (1 + K [Df]) (Eq.24) and [Do] = K° [Df] (Eq.5) w q = Vo/Vw = o i l : w a t e r r a t i o Vo = q Vw S u b s t i t u t e the values of [Db], [Do] and Vo i n Eq.40 and rearrange M - { [ [ • » ] + n K [M] [Df] / (1 + K [D f ] ) j Vw , K° [ D f ] V „ } / (q Vw + Vw) (Eq.41) 42 or | [Df] [ l + n K [M] / (1 + K [Df]) + K° q jj [D] = < [Df ] I 1 + n K [M] / (1 + K [Df]) + f q |} / (q + 1) (Eq.42) For an acid preservative: [Df] = [D f] (1 + Ka/[H +]) where [D^] i s the concentration of unionized preservative i n the aqueous phase, Ka i s the ionization constant, [H +] i s the hydrogen ion concentra-tion and K° = [Do]/[Df] = [Do]/<[D;](l + Ka/[H']) | DJ] (1 a/[H +]) j K° (1 + Ka/[H +]) = [Do]/[Di] = K'° w i . w where K'° i s the pH independent, concentration dependent d i s t r i b u t i o n c o e f f i c i e n t . Substituting the values of K° and [Df] i n Eq.42 gives: [D] = |[D£] (1 + Ka/[H +]) [ 1 + n K M / C l + K [D f] (1 + Ka/[H +])) + K'° q / ( l + Ka/[H +]) J j /(q + 1) (Eq.43) Equations similar to 42 and 43 have been derived by other authors: (i) Kostenbauder (1962); Patel and Romanowski (1970). W = [Df ] ^  R Vw + K° V o | (Eq.44) and for acid preservative, W = [Df]{| R + Ka/[H7] | Vw + K" VO } (Eq.45) ( i i ) Garrett (1966). | | \ [H + jvw + K° VoJ> r m [D] = [D^] . f l . f2 .. f3 = [ D J < 1 + ) n i [Mi]/ j^Ki+[D f] (1 + Ka/[H +] + Kw^jjl 1 + + K°<l} (Eq.46) 43 where f l is the binding enhancement factor, f2 is the oil-water d i s t r i -bution and ionization enhancement factor, f3 is the instability enhance-ment factor, K r^ is the intrinsic dissociation constant for preservative-surfactant complex, k' is the 1st order rate constant for the decomposition of preservative and t is the time required for decomposition, ( i i i ) Bean, Konning and Malcolm (1969). [D] = [ D f ] | R + K°qj /'(q + 1) (Eq.47) Basically, a l l these equations are similar except for the binding parameters used. In the equation of Kostenbauder (1962), Bean, Konning and Malcolm (1969) and Patel and Romanowski (1970), the term 1 + n K [M] / (1 + K[D f]) of Eq.42 is replaced by R, where R = 1 + n K [ M ] , i.e., for a given macromolecule concentration the R value, or binding or solubilization constant, was assumed to be independent of [D^]. As has been described elsewhere (Kazmi and Mitchell, 1971a), this i s true only in special cases (see page 167>)=. In Garrett's equation (1966) the term 1 + n'K [M] / (1 + K [D f]) is replaced by 1 + n [M] / (K' + [D f]) where K' is the intrinsic dissociation'constant and is equal to 1/K. Garrett (1966) also takes into account an instability factor where the preservative degrades by 1st order kinetics. This correction is seldom necessary because most of the commonly used preservatives are stable under the conditions of use., ; For an emulsion containing a surfactant with two classes of binding sites, Eq.42 and 43 can be written: [D] I J- I -LA A A, 4. „ n / (q + 1) , (Eq.48) = i [ D f ] 1 + n ^ M / (1 + K ^ ] + n 2K 2[M] / (1 + K 2[D f]) 1} + K°q wn 44 [ D ] = | [ D p (1 + Ka/[H +]) |^  1 + n ^ M / C l + K ^ D ' ] (1 + Ka/[H +])) + n 2K 2[M]/(l + K 2 [ D p (1 + Ka/[H +])) + K'°q / ( 1 + Ka/[H +]) J j /(q + 1) (Eq . 4 9 ) H. Methodology (a) D i s t r i b u t i o n of preservatives i n oil-water systems Methods for the determination'of the oil-water p a r t i t i o n c o e f f i c i e n t of preservatives and drugs can be divided into two categories, (I) the shake-out method, r e s u l t i n g in.the formation of oil-water emulsions and (II) methods where emulsion formation i s avoided. (I) - The shake-out methods: In these methods (Garrett and Woods, 1 9 5 3 ; Hibbott and Monks, 1 9 6 1 ; Bean and Heman-Ackah, 1 9 6 4 ; Anderson and Cho, 1 9 6 7 ; Bean, Konning and Malcolm, 1 9 6 9 ) the oil-water mixture with preser-vative or drug i s shaken at constant temperature u n t i l equilibrium i s attained. The mixture i s allowed to stand and the oil-water phases are separated and analyzed f o r preservative. An inherent problem associated with t h i s method i s that of e m u l s i f i c a t i o n or sometimes dispersion of f i n e droplets of one phase into the other phase. As a r e s u l t complete separation of the two phases becomes very d i f f i c u l t . A l l e n and McDowell ( 1 9 6 0 ) reported that shake-out methods can also r e s u l t i n anomalous e q u i l i b r i a . In s p i t e of these drawbacks, these methods are popular because of convenience and compared with non-shake-out methods l e s s time i s required f o r equilibrium to be attained. Where e m u l s i f i c a t i o n i s a problem, separation of the o i l -water phases can be achieved by u l t r a c e n t r i f u g a t i o n (Garrett, 1 9 6 2 ) . 45 (II) - Other methods: To avoid e m u l s i f i c a t i o n , other methods have been devised for the determination of the oil-water p a r t i t i o n c o e f f i c i e n t of preservatives and drugs. P a t e l and Romanowski (1969) determined p a r t i t i o n c o e f f i c i e n t s using a two-chambered d i a l y s i s technique, with o i l i n one compartment and water i n the other. The two compartments were separated by a semipermeable membrane, permeable only to preservative molecules. They found close agreement between p a r t i t i o n c o e f f i c i e n t s determined by the d i a l y s i s technique and those determined by shake-out method.. Reese et a l . (1964) and D o l u i s i o and Swintosky (1964) developed a simple rocking apparatus for routine determination of d i s t r i b u t i o n c o e f f i c i e n t s . With t h i s apparatus, up to 36 two-phase samples i n c y l i n d r i c a l tubes were equi-l i b r a t e d by rocking the h o r i z o n t a l tubes at one cycle per minute through an arc of 45°. This rocking causes the i n t e r f a c e between the two immiscible phases to expand and contract slowly. I t also causes the shape of each phase to vary constantly. These two actions f a c i l i t a t e uniform d i s t r i b u t i o n of solute within each phase and f a c i l i t a t e drug transfer from one phase to the other. Emulsion formation i s n e g l i g i b l e since l i t t l e turbulence i s created. The authors found good agreementbbetween r e s u l t s obtained by t h i s method and r e s u l t s from shake-out methods. D i s t r i b u t i o n c o e f f i c i e n t s of some drugs sparingly soluble i n the aqueous phase has also been determined by paper chromatography (Bowen, James and Roberts, 1970). (b) Interaction of preservatives with nonionic surfactants Various methods used for assessing preservative-surfactant i n t e r a c t i o n have been reviewed elsewhere CWedderburn, 1964; Parker and Barnes, 1967; Elworthy, Florence and Macfarlane, 1968). These methods 46 f a l l into two groups: (I) physico-chemical methods and (II) biological methods. (I) Physico-chemical methods: These methods can be divided into two categories. The f i r s t groupadepends upon the properties of the interacting molecule; the second, on the behaviorrof the macromolecule. Methods depending on the properties of the interacting molecule include solubility analysis (Patel and Kosten-bauder, 1958; Blaug and Ahsan, 1961; Goodhart and Martin, 1962; Lundi and Held, 1966; Mitchell and Brown, 1966; Humphreys and Rhodes, 1968; Kazmi and Mitchell, 1971, and others), equilibrium dialysis (Patel and Kostenbauder, 1958; Patel and Foss, 1964, 1965; Breuninger and Goettsch, 1965; Mitchell and Brown, 1966; Anderson and Morgan, 1966; Kazmi and Mitchell, 1971, 1973, and many others), dynamic dialysis (Ikeda et a l . , 1971; Crook and Brown, 1973), turbidimetric t i t r a t i o n (a. visual: Higuchi and Lach, 1954; Guttman and Higuchi, 1956. b. photometric: Kabadi and Hammarlund, 1966), potentiometric ti t r a t i o n (Donbrow and Rhodes, 1963a, 1964, 1965; Evans, 1964, 1966; Donbrow and Jacobs, 1966), pH measurement (Mitchell and Brown, 1966), molecular sieve technique (Ashworth and Heard, 1966; Donbrow, Azaz and Hamburger, 1970), etc. Methods depending on the behavior of the macromolecule include differential interference refractometry (Choulis, 1970; Choulis and Rhodes, 1970), surface tension (Horin and Arai, 1970), viscometry (Horin and Arai, 1970), density measurements (Harkins et a l . , 1946), X-ray diffraction (Harkins et a l . , 1946). Ultraviolet spectroscopy (Riegelman et: a l . , 1958; Anderson and Slade, 1965; Donbrow and Rhodes, 1966; Kabadi and Hammarlund, 1966; Corby and Elworthy, 1971a,b; Choulis, 47 1973) and N M R spectroscopy (Donbrow and Rhodes, 1966, 1967; Jacobs et a l . , 1971; Corby and Elworthy, 1971a,b) have al s o been used to study p r e s e r -v a t i v e - s u r f a c t a n t i n t e r a c t i o n by comparing the spectrum of p r e s e r v a t i v e i n the presence of s u r f a c t a n t . E q u i l i b r i u m d i a l y s i s and e q u i l i b r i u m u l t r a -f i l t r a t i o n techniques were used f o r studying p r e s e r v a t i v e - s u r f a c t a n t i n t e r -a c t i o n i n the present work. Therefore, these two techniques w i l l be discussed i n m o r e . d e t a i l . ( i ) E q u i l i b r i u m d i a l y s i s ; I n t e r a c t i o n s between p r e s e r v a t i v e s and s u r f a c t a n t s may be s t u d i e d q u a n t i t a t i v e l y using the e q u i l i b r i u m d i a l y s i s technique. A container i s d i v i d e d i n t o two compartments by a semipermeable membrane. A s u r f a c t a n t s o l u t i o n w i t h p r e s e r v a t i v e i s placed i n one compartment and aqueous s o l u t i o n w i t h p r e s e r v a t i v e i s placed i n the other compartment. The semipermeable membrane should be permeable only to p r e s e r v a t i v e molecules but not to s u r f a c t a n t monomer. At e q u i l i b r i u m , the t o t a l number of p r e s e r v a t i v e molecules i n the s u r f a c t a n t compartment w i l l exceed that i n the aqueous compartment. The d i f f e r e n c e between the concen-t r a t i o n s i n the two compartments i s a measure of [Db], Two p o s s i b l e sources of e r r o r , namely the Donnan e f f e c t and membrane bi n d i n g of p r e s e r v a t i v e s , must be considered before applying t h i s technique. i When a charged macromolecule, [ M ] , i s r e t a i n e d i n one of the two compartments, at e q u i l i b r i u m , the c o n c e n t r a t i o n of d i f f u s i b l e ions i s no longer i d e n t i c a l across the membrane. This phenomenon i s described as the Donnan e q u i l i b r i u m (Overbeek, 1956). In d i l u t e s o l u t i o n of macromolecule, the Donnan e f f e c t can be neglected only i f the c o n c e n t r a t i o n of the d i f f u s i b l e ions i s ; reasonably high and the valency of the macromolecule i s f a i r l y low. In a solvent system of high i o n i c s t r e n g t h and pH at which the macromolecule 48 has a small valency charge, the abnormal d i s t r i b u t i o n of small molecules across the membrane due to Donnan equilibrium can be neglected. The d i a l y s i s membrane may act as a binding s i t e for the preser-vative molecule (Patel and Kostenbauder, 1958; Kazmi, 1971) and a c o r r e c t i o n must be made for t h i s i n t e r a c t i o n . Corrections are generally made by using a c o n t r o l i n which no macromolecule i s present.. I t i s then possible to measure the " l o s s " of small molecule from one compartment to the other across the semipermeable membrane. It has been observed that the extent of membrane binding i s proportional to the amount of preser-vative added to the system (Patel and Kostenbauder, 1958; Schoenwald and Belcastro, 1969; P a t e l arid Nagabhushan, 1970; Kazmi, 1971). The main advantage of t h i s method i s that an i n t e r a c t i o n can be studied through a range of free preservative concentration [Df] and i n t h i s way i t i s possible to cover a wide range of " r " values (see Eq.24). Thus, more information about the i n t e r a c t i o n can be obtained than with the commonly used s o l u b i l i t y method. The s o l u b i l i t y method i s a "one point method" i n which [Df] i s constant for a l l surfactant concentrations, and therefore only one value of " r " i s obtained (see Eq.24). Techniques employed to carry out equilibrium d i a l y s i s have varied from a d i a l y s i s bag placed i n a b o t t l e (Deluca and Kostenbauder, 1960) to two-chambered d i a l y s i s c e l l s with a semipermeable membrane separating the two compartments (Patel and Foss, 1964). The main advantage of c e l l s over a bag i s that a better c o n t r o l of membrane binding i s attained because the surface area of the membrane remains more or le s s constant throughout the study. 49 Various.types of d i a l y s i s membranes are described by Craig (1965). Kostenbauder elt a l . (1969) discussed the use of nylon membrane i n d i a l y s i s studies. Nylon membrane reacts with phenolic compounds (Patel and Kosten-bauder, 1958; P a t e l and Foss, 1964). Hence, rubber membranes have been used i n the study of the i n t e r a c t i o n of phenolic preservatives with surfactants (Patel and Foss, 1964; M i t c h e l l and Brown, 1966; P a t e l , 1967). Cellophane membranes are widely used i n equilibrium (Chakravarty and Lach, 1959; Breuninger and Goettsch, 1965; Kabadi and Hammarlund, 1966; Matsumoto et a l . , 1966;. P a t e l , 1967; Patel and Romanowski, 1970; Ikeda et a l . , 1971) and dynamic (Matsumoto et a l . , 1966; Matsumoto, 1966; Ikeda et_ a l . , 1971) d i a l y s i s studies involving the i n t e r a c t i o n of preser-vatives and drugs with nonionic surfactants. Cellophane has also been used to study the e f f e c t s of surfactants on the d i f f u s i o n of drugs across membranes (Matsumoto et a l . , 1966; Matsumoto, 1966; Short et a l . , 1970; Short and Rhodes, 1972; Withington and C o l l e t , 1972; C o l l e t and Withington 1973). I d e a l l y , such in v e s t i g a t i o n s require that the membrane be impermeabl to the nonionic surfactant while allowing d i f f u s i o n of the drug and that the osmotic d i f f e r e n t i a l across the membrane i s n e g l i g i b l e . Thereeisdsomeicontroversy concerning the permeability of c e l l o -phane d i a l y s i s membranes to nonionic surfactants. Patel and Kostenbauder (1958) reported that Visking cellophane membrane was permeable to polysorbat 80, and they considered t h i s membrane unsatisfactory for equilibrium d i a l y s i s work. This observation was supported by Nishida et a l . (1964). Breuninger and Goettsch (1965) found that, although Visking cellophane membrane was permeable to polysorbate 80, Fisher cellophane membrane was 50 impermeable to the same surfactant. Matsumoto et^ a l . (1966) studied the permeability of polysorbate 80 and a polyoxyethylene lauryl ether through Visking cellophane membrane, using dynamic dialysis (without stirring the solutions) under sink and nonsink conditions. They found that Visking cellophane membrane was practically impermeable to the nonionic surfactants and that only impurities such as low molecular weight polyethylene glycols, r passed through i t . Patel (1967) compared Fisher cellophane membrane with nylon and rubber membranes in equilibrium dialysis studies, involving the interaction of several preservatives with cetomacrogol and polysorbate 80. Since close agreement was found between data obtained using Fisher cellophane membrane and those obtained using the rubber or nylon membranes, i t was assumed that the cellophane membrane was impermeable to cetomacrogol and polysorbate 80. Ikeda e_t a l . (1971) used Visking cellophane membrane in equilibrium and dynamic dialysis techniques to study the interaction of barbiturates with a polyoxyethylene ether surfactant. Although thessurfactant permeated through the cellophane membrane, the amount passed during 48 hours was below the CMC of the surfactant. Short et_ a l . (1970) reported that Visking cellophane membrane was impermeable to Texofor A30, 45 and 60 and that only small traces of nonsurface-active impurities passed through i t . Although the permeability of cellophane to polyethylene glycols has been studied less extensively thaa the nonionic surfactants, i t has been shown that cellophane i s permeable to polyethylene glycols with a molecular weight less than 20,000 (Shaffer et a l . , 1950; Kabadi and Hammarlund, 1966). 51 Polyethylene g l y c o l s permeate cellophane membranes more r e a d i l y than nonionic surfactants of s i m i l a r molecular weight. Rubber ( M i t c h e l l and Brown, 1966) and nylon (Kazmi and M i t c h e l l , 1970) membranes were used i n equilibrium d i a l y s i s studies of the i n t e r a c t i o n of various preservatives with the nonionic surfactant cetomacrogol, since q u a l i t a t i v e tests showed that cellophane i s permeable to the nonionic surfactant and that volume changes occurred as a r e s u l t of osmosis. Apart from Matsumoto et a l . (1966), few workers using cellophane appear to have corrected f o r the volume changes. In view of the continued use of cellophane, i t seemed desirable as part of t h i s work to make quantitative measurements of the permeability of cellophane membranes to cetomacrogol and to assess the ef f e c t s of surfactant permeation and osmosis on the binding constants for the i n t e r a c t i o n between the surfactant and a preservative determined using the equilibrium d i a l y s i s technique. ( i i ) D i a f i l t r a t i o n (or Equilibrium U l t r a f i l t r a t i o n ) : Various methods have been used to study i n t e r a c t i o n between preservatives and nonionic surfactants. Most of these methods are slow and require numerous separate experiments to f u l l y characterize the i n t e r a c t i o n . Thus, there was s t i l l need of a simple, rapid and r e l i a b l e means of evaluating binding parameters. The d i a f i l t r a t i o n technique appeared to answer these needs. Therefore, an attempt was made to evaluate d i a f i l t r a t i o n technique for studying preservative - surfactant i n t e r a c t i o n . In the present i n v e s t i g a t i o n , a comparison has been made between the binding parameters obtained using d i a f i l t r a t i o n and equilibrium d i a l y s i s techniques. D i a f i l t r a t i o n i s a r e l a t i v e l y new technique which has been used to study drug-protein i n t e r a c t i o n s ( B l a t t , Robinson and B i x l e r , ,1968; Farese, 52 Mager and B l a t t , 1970; Ryan and Hanna, 1971; Crawford, Jones, Thompson and W e l l s , 1972; Palmer, 1972; Barnes, M i t c h e l l , Palmer and Pernarowski, 1973; Campion and Olsen, 1974). The experimental procedure can be best described w i t h reference to F i g . 3. S o l u t i o n of drug i n water or b u f f e r i n the r e s e r v o i r i s forced under pressure i n t o the u l t r a f i l t r a t i o n c e l l c o n t a i n i n g the s o l u t i o n of macromolecule. The macromolecule-free u l t r a f i l t r a t e passes through the membrane and i s d i r e c t e d to a f r a c t i o n c o l l e c t o r . U l t r a f i l t r a t e i s c o l l e c t e d u n t i l the e f f l u e n t drug i s i n e q u i l i b r i u m w i t h the f r e e drug i n the c e l l which i s equal to the feed c o n c e n t r a t i o n . Drug bound to macro-molecule i s i n e q u i l i b r i u m w i t h the f r e e drug i n the c e l l which i s equal to the co n c e n t r a t i o n i n the u l t r a f i l t r a t e . Hence, a n a l y s i s of the drug c o n c e n t r a t i o n i n the u l t r a f i l t r a t e permits c a l c u l a t i o n of the f r a c t i o n of drug bound to macromolecule from zero f r e e drug c o n c e n t r a t i o n to that of the feed s o l u t i o n s . The r e s u l t s f o r bound and f r e e drug can be t r e a t e d i n a number of ways to o b t a i n values f o r the a s s o c i a t i o n constant, K, and number of b i n d i n g s i t e s , n ( B l a t t , Robinson and B i x l e r , 1968; Ryan and Hanna, 1971). The d i a f i l t r a t i o n technique appears to o f f e r s e v e r a l advantages over commonly used techniques such as e q u i l i b r i u m d i a l y s i s and g e l f i l t r a t i o n etc;. 1. This technique enables a complete b i n d i n g curve to be determined from a s i n g l e experiment l a s t i n g a few hours. 2. This technique lends i t s e l f to complete automation and computer a n a l y s i s of data. A n a l y s i s Figure 3. E q u i l i b r i u m U l t r a f i l t r a t i o n Apparatus A. C/D S e l e c t o r B. Res e r v o i r c o n t a i n i n g feed s o l u t i o n C. U l t r a f i l t r a t i o n c e l l D. S t i r r i n g bar E. Magnetic s t i r r e r F. . Membrane. Ln G. F r a c t i o n c o l l e c t o r . <-° H. Temperature c o n t r o l l e d water-baths 54 3. The b i n d i n g parameters d e s c r i b i n g the i n t e r a c t i o n are r e a d i l y determined from the b i n d i n g curve. I I B i o l o g i c a l methods As discussed e a r l i e r , physico-chemical methods have been used e x t e n s i v e l y f o r studying p r e s e r v a t i v e - s u r f a c t a n t i n t e r a c t i o n s because these methods are l e s s time consuming, q u a n t i t a t i v e i n nature, and give i n f o r m a t i o n regarding the mechanism of i n t e r a c t i o n . However, these methods do not take i n t o account the v a r i o u s b i o l o g i c a l v a r i a b l e s a f f e c t i n g m i c r o b i a l growth. Hence, b i o l o g i c a l methods are o f t e n employed f o r e v a l u a t i n g p r e s e r v a t i v e e f f i c a c y i n a system. However, compared w i t h physico-chemical methods, these methods are l a b o r i o u s , time consuming, o f t e n q u a l i t a t i v e i n nature, and give no i n f o r m a t i o n about the mechanism of i n a c t i v a t i o n . A number of authors ( A l l a w a l a and Riegelman^i 1953; Pisano and Kostenbauder, 1959; Blaug and Ahsan, 1961a; M i t c h e l l , 1964; Evans and Dunbar, 1965; Anderson and Morgan, 1966; P a t e l , 1967; Humphrey, Richardson and Rhodes, 1968; Henderson and Newton, 1969; Bradshaw, Rhodes and Richardson, 1972) have attempted to c o r r e l a t e the data obtained from physico-chemical s t u d i e s w i t h the b i o l o g i c a l a c t i v i t y of such systems. I n v e s t i g a t i o n s of t h i s type are important because they give i n f o r m a t i o n about both physico-chemical as w e l l as b i o l o g i c a l f a c t o r s and should save a tremendous amount of time by a v o i d i n g much ' t r i a l and e r r o r ' f o r m u l a t i o n which i s common where pur e l y b i o l o g i c a l methods are employed f o r evaluating, p r e s e r v a t i v e e f f i c a c y . B i o l o g i c a l a c t i v i t i e s can be evaluated e i t h e r by measuring the absolute a n t i m i c r o b i a l a c t i v i t y of each system, or some b i o l o g i c a l response which p a r a l l e l s the p r e s e r v a t i v e a c t i o n on m i c r o b i a l c e l l s . A b r i e f 55 d i s c u s s i o n of the v a r i o u s methods used fori e v a l u a t i n g the a n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s i n s u r f a c t a n t systems f o l l o w s . ( i ) Basic m i c r o b i o l o g i c a l method: The simplest m i c r o b i o l o g i c a l technique f o r demonstrating the antagonism between a p r e s e r v a t i v e and a s u r f a c t a n t i s to compare the growth of t e s t organismsoon/.in a s u i t a b l e growth promoting media c o n t a i n i n g p r e s e r v a t i v e i n the presence and absence of s u r f a c t a n t . The c r i t e r i o n chosen to assess m i c r o b i a l p r o l i f e r a t i o n v a r i e s from v a r i o u s q u a l i t a t i v e t e s t s , such as v i s u a l growth (Wedderburn, 1958), to q u a n t i t a t i v e methods, such as weighing of mycelium ( B o l l e and Mirimanoff, 1950; de Navarre and B a i l e y , 1956; de Navarre, 1957). B o l l e and Mirimanoff (1950) i n v e s t i g a t e d i n t e r a c t i o n s between seven nonionic s u r f a c t a n t s and three p r e s e r v a t i v e s u s i n g P e n i c i l l i u m spp. and A s p e r g i l l u s n i g e r as t e s t organisms. Into 50 ml f l a s k s c o n t a i n i n g 10 ml of Jaag medium was added e i t h e r (a) a standard suspension of spores, (b) spores and p r e s e r v a t i v e i n p r o g r e s s i v e d i l u t i o n , (c) spores and s u r f a c t a n t , or (d) a mixture of spores, p r e s e r v a t i v e and s u r f a c t a n t . The clumps of mycelium were weighed a f t e r 10 days of i n c u b a t i o n at 28°. S i m i l a r methods have been used by other workers (de Navarre and B a i l e y , 1956; de Navarre, > 1957; ...Barrand T i c e , 1957; Wedderburn, 1958). i ( i i ) Minimum i n h i b i t o r y c o n c e n t r a t i o n t e s t s : The e f f e c t of a s u r f a c t a n t on the b a c t e r i o s t a t i c a c t i v i t y of a p r e s e r v a t i v e can be evaluated by d e t e r -mining the minimum i n h i b i t o r y c o n c e n t r a t i o n (MIC) values against a range of organisms i n the presence and absence of a s u r f a c t a n t . E s s e n t i a l l y a MIC experiment i n v o l v e s s e t t i n g up a s e r i e s of p l a t e s or tubes c o n t a i n i n g c u l t u r e medium, the t e s t organism and i n c r e a s i n g concentrations of the p r e s e r v a t i v e . 56 The MIC i s the l e a s t c o n c e n t r a t i o n of the p r e s e r v a t i v e found to i n h i b i t growth. Beckett et_ al_. (1959) demonstrated i n h i b i t i o n of b a c t e r i o s t a t i c a c t i v i t y of h e x y l r e s o r c i n o l against E. c o l i by cetomacrogol, using a MIC t e s t . Pisano and Kostenbauder (1959) obtained MIC values of methyl paraben against Aerobacter aerogenes and A s p e r g i l l u s n i g e r i n the presence of polysorbate 80. The increased amount of paraben r e q u i r e d was compared w i t h the c o n c e n t r a t i o n p r e d i c t e d by e q u i l i b r i u m d i a l y s i s s t u d i e s . S i m i l a r s t u d i e s have been c a r r i e d out by other authors using f u n g i (Blaug and Ahsan, 1961a; Matsumoto and A o k i , 1962; P a t e l , 1967), and b a c t e r i a (Blaug and Ahsan, 19,61a; Evans and Dunbar, 1965) as t e s t organisms. Since t h i s technique i n v o l v e s a m i c r o b i o s t a t i c end p o i n t i t i s not s u i t a b l e f o r determining c o n c e n t r a t i o n r e q u i r e d to produce s t e r i l i t y . I Further l i m i t a t i o n s of the use of a MIC are discussed on page 174. ( i i i ) I n h i b i t i o n zone techniques: I n h i b i t i o n zone techniques such as the cup-plate, c y l i n d e r - p l a t e , paper d i s c - p l a t e , d i t c h - p l a t e and wheel-plate methods r e l y on the d i f f u s i o n of p r e s e r v a t i v e from a c e n t r a l r e s e r v o i r i n t o the surrounding medium. Test organisms surrounding the r e s e r v o i r w i l l be i n h i b i t e d , r e s u l t i n g i n a zone of no growth around the r e s e r v o i r . By comparing zones produced i n the presence and absence of macromolecule i n the r e s e r v o i r , i n f o r m a t i o n may be obtained concerning the e f f e c t of the macromolecule on d i f f u s i o n p r o p e r t i e s of p r e s e r v a t i v e . Agar d i f f u s i o n t e s t s are q u i t e u n s a t i s f a c t o r y f o r e v a l u a t i n g the degree of i n t e r a c t i o n between p r e s e r v a t i v e s and s u r f a c t a n t s . D i f f u s i o n c h a r a c t e r i s t i c of the p r e s e r v a t i v e i n agar i s not only a f f e c t e d by 57 preservative-surfactant i n t e r a c t i o n , but other factors may also be involved. Several authors (Sherwood, 1942; Quisno et a l . , 1946; Foter and Nisonger, 1950) have demonstrated that agar reduces the germicidal potency of quaternary ammonium compounds (quats) due to physi c a l adsorption. Thus i n t e r a c t i o n of the preservative with agar w i l l r e s u l t i n a reduced zone of i n h i b i t i o n , and hence r e s u l t s derived from the measurements of the zones w i l l provide a f a l s e information regarding preservative-surfactant binding. I t i s i n t e r e s t -ing to note that the few authors whose conclusions on the e f f e c t s of nonionic surfactants on preservatives d i f f e r from the generally accepted views on th i s topic used these kinds of techniques. Schwarz and Levy (1957) used the agar cup-plate technique i n a study of the compatibility of c e r t a i n preser-vatives, including quats with carbopol 934. They noted no marked decrease i n the a c t i v i t y of any of the preservatives, since i n a c t i v a t i o n of c a t i o n i c preservatives by the anionic carbopol had been expected. Agar plate d i f f u s i o n techniques have given r e s u l t s suggesting that surface-active agents enhance the a n t i b a c t e r i a l a c t i v i t y of hexachlorophene (Gregg and Zopf, 1951). Anderson and Morgan (1966) used the paper d i s c - p l a t e method to study i n t e r -a ction of hexachlorophene with a number of nonionic surfactants. No c o r r e l a t i o n was obtained between t h e i r physico-chemical and mi c r o b i o l o g i c a l r e s u l t s . (iv) T u r b i d i t y methods: The growth rate of micro-organisms i n broth may be monitored by measuring the change i n t u r b i d i t y with time. The growth rate i s characterized by an i n i t i a l lag phase followed by a logarithmic growth phase where on a s t a t i s t i c a l basis each v i a b l e c e l l i s reproducing at the same rate as a l l the others,(Brown and Garrett, 1964). Brown and Richards (1964a,b) measured the e f f e c t of several compounds oh the growth rate of exponentially d i v i d i n g c e l l s of IS. c o l i . They found (1964b)"that 58 the a n t i b a c t e r i a l a c t i v i t y of polymixin-B s u l f a t e , benzalkonium chloride and chlorhexidine against P_. aeruginosa was s u b s t a n t i a l l y increased i n the presence of a low concentration of polysorbate 80. The cultures of P_. aeruginosa i n nutrient broth were measured i n the logarithmic phase of growth spectrophotometrically at 420 nm. Richards and Hardie (1972) used a s i m i l a r technique to study the e f f e c t of polysorbate 80 and phenylethanol on the a n t i m i c r o b i a l a c t i v i t y of f e n t i c h l o r , Polysorbate 80 antagonised the a c t i v i t y of f e n t i c h l o r against exponential phase cultures of S_. aureus » Pr. v u l g a r i s , and IS. c o l i , but phenylethanol enhanced the a c t i v i t y of f e n t i c h l o r against s i m i l a r cultures of E_. c o l i and Pr. v u l g a r i s . Brown (1966) described a rapi d , economical method of evaluating antimicrobial a c t i v i t y employing turbidimetric measurements, and a s i m i l a r technique has been used"'by other workers (Hugo and Foster, 1964). In most of the test methods mentioned so f a r , the b i o l o g i c a l a c t i v i t y of the preservative-macromolecule system i s determined i n the presence of the culture media. This i s a serious l i m i t a t i o n because i t has been shown (Pisano and Kostenbauder, 1959; Blaug and Ahsan, 1961a) that culture media are capable of a l t e r i n g the i n t e r a c t i o n of preservatives with macromolecules so that s i g n i f i c a n t l y more or l e s s preservative i s bound. Thus, v a l i d comparison of the m i c r o b i o l o g i c a l data can be made only i f the binding c h a r a c t e r i s t i c s f or the combined macromolecule culture medium system are known. (v) Survivor-concentration-time curve technique: M i c r o b i o c i d a l a c t i v i t y may be determined by inoculating a s o l u t i o n of preservative with a known number of test organisms and subsequently carrying out v i a b l e counts. Ei t h e r the preservative concentration i s kept constant and v i a b l e counts 59 made a f t e r increasing time periods (Eisman et a l . , 1957; Hugo and Newton, 1964; Marx et a l . , 1968), or increasing concentrations of preservative are tested and v i a b l e counts performed a f t e r a fix e d period of time (Hugo and Newton, 1964; Humphreys et a l . , 1968; Henderson and Newton, 1969; Bradshaw eit a l . , 1972). The method employed depends on which parameter, s t e r i l i z i n g time or preservative concentration, i s the more important. Experiments may bet-performed i n the presence of macromolecules to determine t h e i r e f f e c t on b a c t e r i c i d a l a c t i v i t y . Marx et a l . (1968) studied the behavior of a n t i b a c t e r i a l agents i n solutions of nonionic surfactants using v i a b l e count technique. Surfactant solutions containing varying concentrations of a preservative were inoculated with a given number of test organisms and the death rate was followed over a period of several weeks. Surfactant solutions containing preservative showed a steady decline i n the microbial count. However, the unpreserved surfactant solutions used as controls showed and increase i n the microbial count. Similar techniques have been used by other workers to study the e f f e c t of macromolecules on the a n t i b a c t e r i a l a c t i v i t y of preservatives (Eisman et a l . , 1957; Taub et a l . , 1958; Richardson and Woodford, 1964). (vi) E x t i n c t i o n time methods: An ex t i n c t i o n time method for evaluating b a c t e r i c i d a l a c t i v i t y was described by Cook and W i l l s (1954) and Berry and Bean (1954). E s s e n t i a l l y the method involves: (a) introducing a small volume of preservative s o l u t i o n into each of a r e p l i c a t e s e r i e s of tubes i n a constant temperature water bath; (b) inoculating each tube with a constant number of test organisms; (c) quenching the k i l l i n g process a f t e r a s u i t a b l e time i n t e r v a l by flooding the tube contents with broth; (d) r e -peating the whole procedure for several d i f f e r e n t exposure times; (e) r e -60 cording the number of negative growth tubes a f t e r incubation for each exposure time; and (f) determining the e x t i n c t i o n time by s t a t i s t i c a l a n a l y s i s . The analysis of e x t i n c t i o n time data i n bioassays has been described by Mather (1949). The e f f i c a c y of a preservative i n the presence of a macromolecule can be evaluated by comparing the e x t i n c t i o n time i n presence and absence of macromolecule (Bean and Berry, 1951, 1953; Allawala and Riegelman, 1953; Beckett et a l . , 1959; M i t c h e l l , 1964). ( v i i ) Other methods; Methods of measuring b i o l o g i c a l responses, which p a r a l l e l the preservative action on m i c r o b i a l c e l l s , are often used to evaluate preservative e f f i c a c y . Ansel (1965) measured the e f f e c t of poly-ethylene g l y c o l s i n the haemolytic a c t i v i t y of phenol towards rabbit erythrocytes. Judis (1962) examined the protection given by polysorbate 80 to E_. c o l i against chloroxylenol. The release of r a d i o - a c t i v e material from l^C l a b e l l e d c e l l s was measured as an index of c e l l damage. Measurements of changes i n b a c t e r i a l and fungal spore volume have been used to assess the e f f i c a c y of a n t i m i c r o b i a l agents i n aqueous and surfactant systems., (Hitchins et a l . , 1963; Gould, 1964; Barnes and Parker, 1966, 1967; Parker et a l . , 1966, 1968; McCafferty and Parker, 1970). Parker et a l . (1966) used a coulter counter to measure the germination swelling of Trichoderma and s u b t i l i s spores i n various preservative or preservative-polysorbate 80 mixtures. No spore swelling occurred i n phenyl mercuric n i t r a t e or chloro-c r e s o l whether the surfactant was present or not, but i n solutions of parabens, cetrimide and Nipastat greater swelling occurred i n the presence of surfactant. Wailes (1962) and Brown (1968) studied the i n h i b i t i o n of r e s p i r a t i o n of bakers yeast i n preservative and preservative-surfactant mixtures using a Warburg apparatus. Wedderburn (1964) used a s i m i l a r technique using P_. aeruginosa as t e s t organism to evaluate the e f f i c a c y 61 of preservatives i n the presence of surface active agents. (c) D i s t r i b u t i o n of preservatives i n oil-water-surfactant systems In the previous section (&,b),.asa mathematical model (Eq.48) for the preservative d i s t r i b u t i o n i n emulsified systems was derived. There are a few l i m i t a t i o n s to the use of t h i s model. (i) Determination of necessary parameters i n Eq.48 i s a lengthy process, p a r t i c u l a r l y i f , as is~-usual,in most pharmaceutical and cosmetic emulsions, preservative mixtures or more than one type of macromolecule i s present. Moreover, even a s l i g h t change i n the formulation of emulsion necessitates a re-evaluation of the various terms. ( i i ) Existence of l i q u i d c r y s t a l l i n e phases and the presence of reversed micelles i n the o i l phase (Friberg and Mandell, 1970) i n complex emulsions would make the mathematical model d i f f i c u l t or impossible. ( i i i ) In an emulsion, some surfactant i s adsorbed at the oil-water i n t e r f a c e and, depending on i t s o i l s o l u b i l i t y , some p a r t i t i o n s into the o i l phase. Both factors reduce the amount of surfactant a v a i l a b l e f o r i n t e r -a ction with the preservative and a f f e c t the oil-water p a r t i t i o n c o e f f i c i e n t . Where appreciable amounts of surfactant are adsorbed or p a r t i t i o n e d into the o i l , determination of the parameters f o r s u b s t i t u t i o n into Eq.48 becomes d i f f i c u l t . In view of these l i m i t a t i o n s i t i s necessary that the model should be v e r i f i e d experimentally f o r the amount of preservative i n each 'phase' of the emulsion and hence, the t o t a l concentration required to provide desired concentration i n the aqueous phase. 62 Very few techniques are available for a quantitative study of the distribution of a preservative between the different phases of an emulsion system. Garrett (1966) suggested an ultracentrifuge technique for the separation of the various phases of an emulsion and subsequent analysis of each phase for preservative content. The main drawback associated with this technique i s the destruction of emulsion structure which may disturb the equilibrium concentrations of preservative in the various phases of the emulsion. Patel and Romanowski (1970) used an equilibrium dialysis technique to determine the concentration of free preservative in the aqueous phase, [D^], of an emulsion. They uti l i z e d a two-chambered glass dialysis c e l l , with emulsion in one compartment and broth in the other (see page. 58 for the effect of culture media on preservative-surfactant interaction). The two compartments were separated by a membrane permeable to preservative but impermeable to o i l and surfactant. At equilibrium, the concentration of free preservative in the aqueous phase was assumed to be equal on both sides of the membrane. Analysis of the aqueous compartment gave the concentration of free preservative in the aqueous phase of the emulsion. Subtracting the amount of preservative in the aqueous phase from the total amount of preser-vative added to the dialysis c e l l gave the total concentration of preservative in the o i l and surfactant phases ([Do] + [Db]). Thus, with this technique, i t i s not possible to separate the concentrations of preservative in o i l , [Do], and surfactant, [Db], phases. Kazmi and Mitchell (1971) developed a three-chambered dialysis 63 technique. With t h i s i t was p o s s i b l e to measure the amount of pr e s e r -v a t i v e i n each phase of the emulsion, i . e . , [D^], [D t] and [Do], and hence the t o t a l c o n c e n t r a t i o n r e q u i r e d to provide the d e s i r e d c o n c e n t r a t i o n i n the aqueous phase. The technique a l s o d i f f e r e n t i a t e d between f r e e s u r f a c t a n t "->.. • and s u r f a c t a n t adsorbed a t the o i l - w a t e r i n t e r f a c e or p a r t i t i o n e d i n t o the o i l phase. Shimamoto et a l . (1973) used an u l t r a f i l t r a t i o n method f o r measuring f r e e p r e s e r v a t i v e c o n c e n t r a t i o n i n the aqueous phase of o i l - i n -water emulsions. The emulsion was c e n t r i f u g e d repeatedly to separate excess of the o i l phase. U l t r a f i l t r a t i o n of the t h i n emulsion separated the aqueous phase from the o i l and s u r f a c t a n t phases. The u l t r a f i l t r a t e was analyzed f o r the f r e e p r e s e r v a t i v e c o n c e n t r a t i o n . This technique i s not r e l i a b l e because the procedure i n v o l v e d i n the separation of the aqueous phase not only d i s t u r b s the emulsion s t r u c t u r e but a l s o r e s u l t s i n the formation of emulsions of v a r y i n g o i l - w a t e r r a t i o s . Both e f f e c t s may change the e q u i l i b r i u m concentrations of p r e s e r v a t i v e i n v a r i o u s phases of the emulsion. Polderman (1973) reported a dynamic d i a l y s i s technique f o r measuring the f r e e p r e s e r v a t i v e i n the aqueous phase of v i s c o u s creams. Using t h i s technique i t was p o s s i b l e to determine the f r e e p r e s e r v a t i v e c o n c e n t r a t i o n i n much shor t e r time than i s p o s s i b l e w i t h e q u i l i b r i u m d i a l y s i s . Samples of preserved o i l - w a t e r cream were f i l l e d i n t o cellophane tubes and d i a l y z e d against a s e r i e s of aqueous s o l u t i o n s of i t s pr e s e r -v a t i v e . Based on the o v e r a l l c o n c e n t r a t i o n of the p r e s e r v a t i v e i n the cream and i t s d i s t r i b u t i o n c o e f f i c i e n t , a s e r i e s of aqueous s o l u t i o n s were made, 64 so that the concentrations were both higher and lower than the estimated concentration i n the aqueous phase of the cream. D i a l y s i s i n a l l the tubes was performed under s t r i c t l y i d e n t i c a l conditions such as duration, surface of the membrane, temperature, etc. A f t e r a few hours the concen-t r a t i o n of the preservative was determined i n the d i a l y z a t e and the decrease or increase of preservative concentration (AC ) was p l o t t e d against the s t a r t i n g concentration [ c ] . A s t r a i g h t l i n e was obtained and the point where the l i n e intersected the C-axis was taken as the concentration of free preservative i n the aqueous phase of the cream. The l i n e a r i t y of the l i n e was independent of the concentration of preservative s o l u t i o n under d i a l y s i s . The slope of the graph was dependent on the duration of d i a l y s i s . (d) Antimicrobial a c t i v i t y of preservatives i n oil-water-surfactant systems Methods employed f o r evaluating the e f f i c a c y of preservatives i n emulsified systems can be divided into two types. Type (I) methods -designed to test predictions of a n t i m i c r o b i a l a c t i v i t y based on physico-chemical models. These methods are analogous to methods discussed f o r studying preservative-surfactant i n t e r a c t i o n . (II) Methods used to t e s t the e f f i c a c y of a n t i m i c r o b i a l agents i n the f i n a l formulations. These methods include normal usage t e s t s , challenge tests and capacity t e s t s . Type I methods are l e s s time consuming and provide a f i r s t step i n the formulation of the f i n a l emulsion system. However, the r e l i a b i l i t y of these methods f o r evaluating the e f f i c a c y of preservatives under ac t u a l conditions of use has not been s u f f i c i e n t l y tested up to the present time. Type .II methods, on the other hand, are widely used i n industry. They 65 attempt to simulate conditions in which the f i n a l product w i l l be manufactured and ut i l i z e d . Type I Methods Anderson and Chow (1967) determined the minimum inhibitory concentration (MIC) of benzoic acid against Aspergillus niger spores in emulsified systems. The mycelial growth in the emulsions was examined visually. The concentration of preservative required to prevent fungal growth in half of six or more replicates was chosen as satisfactory means of assessing the fungistatic activity of benzoic acid, and this criterion of assessment was referred to as FC50. Bacteria caused spoilage to an even greater extent than fungi (Wedderburn, 1964), and since their growth in an emulsion cannot be detected visually, the MIC test of Anderson and Chow is not suitable for estimating the efficacy of an antimicrobial agent against bacteria in emulsified systems. Patel and Romanowski (1970) used a dialysis technique for deter-mining minimum inhibitory concentration of preservatives in emulsified systems. The assumption was made that the biologic activity of preservative would parallel the concentration of free preservative in the aqueous phase. Various limitations and weaknesses of this method w i l l be discussed later (page 173). Bean et a l . (1962, 1964, 1965, 1969) employed an extinction time method for studying the effects of oil-water partition coefficient, o i l -water ratio, binding with nonionic surfactants and temperature on the anti-microbial activity of preservatives in simple oil-water emulsions and o i l -water emulsions stabilized with nonionic surfactants. Various disadvantages 66 of t h i s method w i l l be discussed l a t e r (page 175). " Few attempts (Marx et a l . , 1968; Shimamoto et a l . , 1973) have been made to study the death r a t e of micro-organisms i n e m u l s i f i e d systems. The accuracy of these methods.has been l i m i t e d due to d i f f i c u l t i e s i n the i s o l a t i o n of micro-organisms from the emulsion. However,- membrane f i l t r a t i o n techniques have been found very u s e f u l f o r i s o l a t i n g micro-organisms from emulsions and ointment bases ( S o k o l s k i and Chidester, 1964; Buhlmann, 1968; Ko and Vanderwyk, 1968; Hambleton.,and Allwood, 1972; Allwood and Hambleton, 1973). S o k o l s k i and Chidester (1964) demonstrated that the membrane f i l t r a t i o n technique o f f e r e d markedly higher r e c o v e r i e s of v i a b l e micro-organisms from petrolatum-based ointments than other methods i n common use. The advantages of the membrane f i l t r a t i o n technique over commonly used i s o l a t i n g procedures w i l l be discussed f u r t h e r under Results and D i s c u s s i o n . Normal usage t e s t s ^ These t e s t s (Sykes and Smart, 1968) r e q u i r e that the product i s made under normal manufacturing c o n d i t i o n s , thus ensuring that i t i s exposed to the expected contamination hazards, and then stored under v a r i o u s c o n d i t i o n s f o r extended p e r i o d s , such as months and years. At i n t e r v a l s samples are examined m i c r o b i o l o g i c a l l y f o r signs of d e t e r i o r a t i o n and s p o i l a g e . Another method (Parker, 1969) that has been suggested i s d a i l y use of the p r e p a r a t i o n w i t h d a i l y sampling f o r v i a b l e counts. There are c e r t a i n advantages as w e l l as weaknesses a s s o c i a t e d w i t h the type of normal usage t e s t s described above. The main advantage i s that the product i s i n o c u l a t e d w i t h ' n a t u r a l ' organisms which have then every opportunity to adapt themselves to grow i n the p a r t i c u l a r medium. Some of the weaknesses of these t e s t s are, ( i ) i t i s a long process, i n v o l v i n g months and years of storage, ( i i ) the inoculum can never be standardized 67 and i s always v a r i a b l e and indeterminate, ( i i i ) more important, these tests do not measure the l e t h a l properties of the preservative i n the product but only i t s a b i l i t y to prevent microbial growth; such a state i s never a stable one and i t can e a s i l y be upset by small changes i n the environmental conditions, or by the occurrence of more r e s i s t a n t organisms. Challenge tests - These are the most commonly used tests (Rdzok et a l . , 1956; Barr and Tice, 1957; Baker, 1959; Charles and Carter, 1959; Nowak, 1963; Marinaro, 1966; Bean, 1967; Olson, 1967; Sykes and Smart, 1968; Flawn et a l . , 1973) for assessing the l e t h a l a c t i v i t i e s of the product against known micro-organisms. These tests are c a r r i e d out by inoculating the product, usually i n the f i n a l containers, with a given type of micro-organism or a mixture of d i f f e r e n t types of micro-organisms, and determining t h e i r l o s s of v i a b i l i t y over a given period of time. The micro-organisms selected are generally those found to be contaminants i n s i m i l a r types of preparations and important both from c l i n i c a l and pharmaceutical view points. Although challenge tests assess l e t h a l ' a c t i v i t y , are more p o s i t i v e and provide a greater margin of safety, these tests do not take into account the performance of a preservative system under conditions of use i n the f i e l d where pos s i b l y i t w i l l be subjected to various cycles of contamination of new unknown micro-organisms during successive use by the patient, or during handling and transfer to .other containers by the pharmacist. Thus there i s a p o s s i b i l i t y of reduction of preservative potency i n the preparation due to successive contamination and consequent d e t e r i o r a t i o n of the product by m u l t i p l i c a t i o n of more r e s i s t a n t organisms. 68 Capacity tests - Recently these tests have been found very popular and have been used by various authors (Cantor and Shelanski, 1951; P r i c k e t t et a l . , 1961; Bryce.and Smart, 1965; MacRae and Johnson, 1965; Lemmex; 1967; Olsen, 1967; Tenenbaum, 1967; Sykes and Smart, 1968; Barnes and Denton, 1969) for evaluating preservative e f f i c a c y i n various types of formulations, e s p e c i a l l y cosmetic creams and emulsions. The basis of these tests i s to measure the capacity of the preservative system to meet cycles of contamination, as i n d a i l y use, and i s perhaps the nearest approach a v a i l a b l e to a true in-use assessment. In these tests products, containing varying concentrations of preservative, are t i t r a t e d (repeated inoculation) with micro-organisms and sampled at d i f f e r e n t time i n t e r v a l s u n t i l a p o s i t i v e end point i s reached. The time cycle can be varied i n accordance with the type of product and i t s usage. C r i t e r i a f o r assessing the e f f i c a c y of preservative systems using challenge or capacity tests have va r i e d considerably i n the l i t e r a t u r e . Kohn (1963) considers a preservative too slow acting to be used i n ophthalmic solutions i f i t cannot s t e r i l i z e a given Pseudomonas suspension within one hour. The United States Pharmacopoea XVIII (1970) considers that an a n t i -m i c r o b i a l agent i s adequate for use i n a product intended for parenteral or ophthalmic administration i f there i s no s i g n i f i c a n t increase i n the number (5 x 10 /20 ml) of Candida albicans or A s p e r g i l l u s niger organisms, and i f the number of v i a b l e vegetative micro-organisms (E. c o l i ; P_. aeruginosa; S_. aureous) i s reduced to not more than 0.1 percent of the i n i t i a l number (5 x 10 /20 ml) and remains below that l e v e l f or a 7 day period within the 69 28-day t e s t p e r i o d . Bean (1967) suggested a time of two hours i n which the p r e s e r -v a t i v e should be capable of s t e r i l i z i n g v e g e t a t i v e c e l l s introduced i n t o the emulsion. Barnes and Denton (1969) showed from t h e i r c a p a c i t y t e s t s that the standard suggested by Bean was d i f f i c u l t to achieve i n formulations such as creams. According to these authors a p r e s e r v a t i v e should reduce 7 3 v i a b l e organisms at approximately 5 x 10 per g or ml by more than 10 4 (cream and suspension) or 10 ( s o l u t i o n ) i n 48 hours. Tenenbaum (1967) suggested that the s t e r i l i z i n g time should be measured i n days or even weeks, in s t e a d of hours. According to Marinaro (1966), "the p r e s e r v a t i v e a c t i o n i s considered e x c e l l e n t , i f m i c r o b i a l l i f e i s absent or a d r a s t i c r e d u c t i o n i s observed w i t h i n 24 hours or l e s s ; good, i f a s a t i s f a c t o r y response i s obtained w i t h i n one week; f a i r , i f i n two weeks, and questionable i f a response i s observed a f t e r t h i s p e r i o d . " Baker (1959) considers a p r e p a r a t i o n i d e a l l y preserved i f 6 x 10 organisms per g are s t e r i l i z e d w i t h i n 2-4 days, and s a t i s f a c t o r y i f the count i s reduced to a few thousand per gram w i t h i n a p e r i o d of s i x days. 70 EXPERIMENTAL A. Apparatus. (a) Amicon D i a f i l t r a t i o n Apparatus. (b) Amsco M e d a l l i o n S e r i e s Autoclave. (c) Beckman DBGT Double Beam Spectrophotometer, w i t h Beckman 10 i n c h Recorder. (d) Bioquest B i o l o g i c a l Cabinet. (e) Fisher-Brand S t e r i l i z e d Disposable P l a s t i c P e t r i Dishes. ( f ) F i s h e r Colony Counter. (g) Gallenkamp Oven- Model OV-160. (h) H i t a c h i 124 Coleman Double Beam Spectrophotometer w i t h H i t a c h i 165 Recorder. ( i ) Haake R 2^ Thermoregulator, ( j ) Hand Powered Homogenizers ( C e n t r a l S c i e n t i f i c Co.). (k) I n t e r n a t i o n a l C e n t r i f u g e , S i z e 1, Model SBV. (1) Isco F r a c t i o n C o l l e c t o r . (m) Labline/Case I m p e r i a l I I Incubator. (n) Magniwhirl (Blue M), Constant Temperature R e f r i g e r a t e d Shaker Water Bath, (o) M i l l i p o r e 6-Place S t e r i l i t y Test M a n i f o l d , (p) M i l l i p o r e S t e r i f i l A s e p t i c F i l t r a t i o n System, (q) M i l l i p o r e Vacuum Pump. (r) P o l a r i t e r Radiometer PO^ Polarograph w i t h Radiometer Drop L i f e Timer, Type DLT, and F i s h e r Calomel E l e c t r o d e , No. 13-639-51. (s) F i s h Tank, 50 L. 71 (t) Two Chambered P l e x i - G l a s s D i a l y s i s C e l l s , as Described by P a t e l and Foss (1964). (u) Membrane (1) Nylon Membrane (0.0005" t h i c k ; Capran 77; A l l i e d Chemical Corporation.) (2) F i s h e r Cellophane ( D i a l y s i s Tubing; 3.6 cm F l a t Width; F i s h e r S c i e n t i f i c Co.) (3) V i s k i n g Cellophane ( D i a l y s i s Tubing; 2.4 cm F l a t Width: Union Carbide Ltd.) (4) M i l l i p o r e F i l t e r s (HAEG; 0A5jU; 47 mm Diameter? M i l l i p o r e Ltd.) (5) D i a f l o U l t r a f i l t r a t i o n Membranes (UM-05; Amicon CCorporation.) (v) Wang 600 Programmable C a l c u l a t o r . B. M a t e r i a l s (a) P r e s e r v a t i v e s 1. Benzoic A c i d , reagent grade (Fisher S c i e n t i f i c Co.). 2. So r b i c A c i d , reagent grade (Eastman Organic Chemicals). 3. Methyl p-hydroxybenzoate, P r o p y l p-hydroxy benzoate, reagent grade ( B r i t i s h Drug Houses). 4. C h l o r o c r e s o l , reagent grade ( B r i t i s h Drug Houses). 5. C h l o r o x y l e n o l , reagent grade (Eastman Organic Chemicals). 72 Nonionic Surfactants Three n - a l k y l polyoxyethylene surfactants (Glover Chemicals Ltd., Cetomacrogol 1000, BPC, where m may be 15 or 17 and n may be 19 to 23, assuming m = 17 and n = 23, the molecular weight was taken as 1300; Texofor A16, where m = 15 and n =16; Texofor A60, where m = 15 and n = 60. Since Texofor A16 and A60 were from the same batch as used by Simons and Rhodes (1971), the molecular weight of Texofor A16 and A60 was calculated using the mean molecular formula determined by the authors using NMR technique. The mean molecular formula of Texofor A16 was the same as claimed by the manufacturer, i.e., CH3 (CH 2) 1 5 (0CH 2CH 2) 1 6 OH, and thus the calculated value of the mean molecular weight was 946. The mean molecular formula of Texofor A60 was CH 3(CH 2) 1 5 (OCH 2CH 2) 7 7 OH, and the calculated value of the molecular weight was 3630. Poly-sorbate 80 (Atlas Chemical Industries) of the general formula. where nj. + n^ + n^ = n, assuming n = 20, the molecular weight was taken as 1308. 0 II 0 CH„C-C, .,H. 73 (c) L i g h t L i q u i d P a r a f f i n , B.P.C. ( B r i t i s h Drug Houses). (d) Sodium C h l o r i d e , reagent grade ( M a l l i n c k r o d t Chemical Works). (e) Phosphomolybdic A c i d (J.T. Baker Chemical Co.). (f) Barium C h l o r i d e , Analar grade (J.T. Baker Chemical Co.). (g) Nitrogen, G grade. (h) H y d r o c h l o r i c A c i d , reagent grade ( M a l l i n c k r o d t Chemical Works). ( i ) T r y p t i c a s e Soy Broth (BBL, D i v i s i o n of Bioquest). ( j ) T r y p t i c a s e Soy Agar (BBL, D i v i s i o n of Bioquest). (k) Bacto Peptone (Difco L a b o r a t o r i e s ) . (1) Deionized Glass D i s t i l l e d Water, (m) Peptone Water Sodium C h l o r i d e - 8.5 g. Bacto Peptone - 1.0 g. D i s t i l l e d Water, Q.S. - 1 L. C. Temperature Unless otherwise s t a t e d a temperature of 25 ± 0.05° was used i n a l l the s t u d i e s . D. A n a l y s i s of Surface-Active Agents (a) P olarographic a n a l y s i s Q u a n t i t a t i v e a n a l y s i s of cetomacrogol was made using a polarograph. The a n a l y s i s was based on the damping of "the polarographic maxima by su r f a c e -a c t i v e agents (Vavruch, 1950; J e h r i n g , 1966). Potassium c h l o r i d e s o l u t i o n (0.002N) gives a very pronounced oxygen.maximum. A comparison of the heights of the maxima i n the presence and absence of cetomacrogol ( F i g . A) and reference to a c a l i b r a t i o n curve ( F i g . 5) permitted the determination of 74 Fig. 4. Polarographic current voltage curves. (a) oxygen maximum of potassium chloride solution (N/500). (b) suppression of oxygen maximum by surface-active agent, h, height of oxygen maximum. F i g . 5. C a l i b r a t i o n curve for the polarographic determination of cetomacrogol. (Different symbols represent separate experiments.) 76 surfactant down to 1.0 mg 1 ^. Since the method i s not s p e c i f i c f o r cetomacrogol, the r e s u l t s are expressed as apparent cetomacrogol concen-t r a t i o n s . To prepare a c a l i b r a t i o n curve, 100 ml each of 0.02N KC1 s o l u t i o n and 0.5% surfactant s o l u t i o n were prepared i n deionized g l a s s - d i s t i l l e d water. One m i l l i l i t e r of 0.5% surfactant s o l u t i o n was transferred to a 100 ml volumetric f l a s k and made up to volume with d i s t i l l e d water, so that the f i n a l concentration of surfactant was 0.005 %. Five m i l l i l i t e r s each of 0.02N s o l u t i o n was pipeted to eleven 50 ml volumetric f l a s k s and to the f i r s t ten volumetric f l a s k s 1-10 ml of 0.005 % surfactant was added re s p e c t i v e l y . F i n a l l y a l l the volumetric f l a s k s were made up to volume with d i s t i l l e d water. Thus the f i n a l concentration of surfactant i n the f i r s t ten volumetric f l a s k s ranged from 1-10 mg:l 1 r e s p e c t i v e l y and the f i n a l normality of KG1 i n a l l the f l a s k s was 0.002N. A l l eleven solutions were subjected to polarographic analysis and the height of the oxygen maximum was measured f o r each s o l u t i o n . The heights of oxygen maxima of 0.002N KC1 solutions containing various concentrations of surfactant (1-10 mg 1 ^) were subtracted from the height of oxygen maximum of 0.002N KC1 s o l u t i o n r e s p e c t i v e l y . The percentage suppression of oxygen maximum was cal c u l a t e d fo r each s o l u t i o n . The height of oxygen maximum, h, (Fig. 4) depends very much on the conditions of a n a l y s i s , such as atmospheric pressure, temperature, e l e c t r o -l y t e concentration, diameter of c a p i l l a r y , drop time, etc. Since i t i s impossible to con t r o l atmospheric pressure, i t was necessary to plo t a new c a l i b r a t i o n curve each time. 77 (b) Phosphomolybdic a c i d t e s t This method i s based on the p r e c i p i t a t i o n of n o n i o n i c - s u r f a c t a n t s of the ethylene-oxide type by phosphomolybdic a c i d i n barium c h l o r i d e - h y d r o -c h l o r i c a c i d medium ( O l i v e r and Preston, 1949; Stevenson, 1954). To det e c t the presence of nonioni c s u r f a c t a n t s i n the s o l u t i o n of the aqueous compartment of a d i a l y s i s c e l l q u a l i t a t i v e l y , an a l i q u o t of the aqueous s o l u t i o n (0.5 ml) was d i l u t e d w i t h 5 ml of g l a s s d i s t i l l e d water i n a t e s t tube. F i v e drops of h y d r o c h l o r i c a c i d (1:4), 5 drops of barium c h l o r i d e (10% w/v) and f i v e drops of phosphomolybdic a c i d (10% w/v) were added and the t e s t tube was w e l l s t i r r e d using a vo r t e x mixer. The presence of nonionic s u r f a c t a n t was detected by the formation of a y e l l o w i s h or y e l l o w i s h green p r e c i p i t a t e . E. A n a l y s i s of the P r e s e r v a t i v e s i n Aqueous.and Surfactant S o l u t i o n s A l i q u o t s of s o l u t i o n s were a p p r o p r i a t e l y d i l u t e d w i t h water ( c h l o r o c r e s o l ) or 0.01N HCl (benzoic a c i d ) , and the p r e s e r v a t i v e concentra-t i o n s were determined by UV spectrophotometry. The absorbance of c h l o r o c r e s o l and benzoic a c i d were measured at 280 nm, and 230 nm. r e s p e c t i v e l y . Where a comparison was made between the bi n d i n g curves f o r the i n t e r a c t i o n of a p r e s e r v a t i v e w i t h a nonionic s u r f a c t a n t i n the absence and presence of another p r e s e r v a t i v e , the s i n g l e p r e s e r v a t i v e s were analyzed using a UV absorbancy-ratio method. This approach was used i n order to avoid d i f f e r e n c e s i n b i n d i n g curves due to a n a l y t i c a l a r t i f a c t s . ;F. A n a l y s i s of P r e s e r v a t i v e Mixtures i n Aqueous and Surfactant S o l u t i o n s Binary mixtures of p r e s e r v a t i v e s were analyzed using the 78 absorbancy ratio method ("Q" analysis) o£; Pernarowski et a l . (1961a,b). The same terminology and symbology has been observed in this work as used by the authors. (a) The spectral characteristics of the individual preservatives of the binary mixture were determined (Figs. 6, 8, 10 and 12). (b) The isoabsorptive point was located by recording the spectrum of a solution of one preservative relative to a solution of another preservative (i.e., the second solution was the "blank"). The i n i t i a l concentrations of both the preservative solutions were identical. The wavelength at which an absorbancy reading of zero was observed represented an isoabsorptive point. (c) The two wavelengths chosen for the analysis were the isoabsorptive point and the wavelength at which one of the two preservatives exhibited maximum absorption, (d) The "Q0" values (Eq.50) for a number of preservativesmixtures were determined. The absorptivity value (a^) of both preservatives at the iso-absorptive point were also determined. For a given preservative combination, value of a^ for both the preservatives were similar, indicating the exact-ness in the determination of the isoabsorptive wavelength. (e) "Q0" v s fraction of one of the components in the preservative mixture (Fy) was plotted (Eq.50), and the data was subjected to least square f i t t i n g to ascertain the equation of the resulting straight line (Figs. 7, 9, 11 and 13). (f) The numerical constants obtained from the "Q" curve were substituted into the Eq.51 and 52, (g) The concentration of individual preservatives in an unknown binary mixture were calculated by substituting the values of Q Q , and a^ into Eq.51 and 52. QQ = (Qy - Qx) Fy + Qx (Eq.50) A_ Qo - Qx 79 0.4 0.3 w u %• pq Pi o w re 0.2 0.1 0.0 200 220 240 260 WAVELENGTH nm. 280 300 F i g . 6. Spectrophotometry curve f o r benzoic a c i d (• •) and s o r b i c a c i d ( ) i n 0.01N HCl using a H i t a c h i Coleman spectrophotometer. Concentration of benzoic acid, and s o r b i c a c i d = 2.0 mg l - 1 . A b s o r p t i v i t y value o f ' p r e s e r v a t i v e s at i s o a b s o r p t i v e wavelength, 236.4 nm = 73.5. 80 1.1 1.0 *. 0.9 cn CNI 5 0.8 o 0.7 0.6 0.5 40 60 BENZOIC ACID (%) Fig. 7. Q curve for benzoic acid and sorbic acidQs Points experimental, line f i t t e d using least squares method. Slope = 0.641. Intercept = 0.613. Correlation coefficient = 0.999. 81 0.5 0.4 w | 0.3 m Pi o CO m < 0.2 0.1 0.0 '• ~'\ V A \ X J-210 220 230 240 250 260 270 280 290 WAVELENGTH nm. F i g . 8. Spectrophotometric curves f o r c h l o r o c r e s o l ( ) and methyl paraben ( ) i n water using Beckman DBGT spectrophotometer. Concentration of c h l o r o c r e s o l and methyl paraben = 5.0 mg 1 _ 1 . A b s r o p t i v i t y value of p r e s e r v a t i v e s at i s o a b s o r p t i v e wavelength, 236 nm = 30.4. 82 o CO pq <3 0.5 0.3 0.2 r 0.1 L 0.0 F i g . 10. 210 220 230 240 250 260 270 280 290 300 WAVELENGTH nm. Spectrophotometry curves f o r c h l o r o c r e s o l ( ) and propyl paraben (-—• ) i n water using H i t a c h i Coleman spectrophotometer. Concentration of c h l o r o c r e s o l and p r o p y l paraben = 5.0 mg l - i . A b s o r p t i v i t y value of p r e s e r v a t i v e s at i s o a b s o r p t i v e wavelength, 233.2 = 27.0. OO CO 0 20 40 60 80 100 CHLOROCRESOL (%) Fig. 11. Q curve for chlorocresol and propyl paraben. Points experimental, line f i t t e d using least squares method. Slope = 1.318. Intercept = 0.509. Correlation coefficient = 0.999. 85 PQ Pi O CO PQ < 0.4 0.3 0.2 0.1 A s _t- _L 220 230 240 250 260 WAVELENGTH nm. 270 280 290 F i g . 12. Spectrophotometry curve f o r c h l o r o x y l e n o l ( •) and methyl paraben ( ) i n water using H i t a c h i Coleman spectrophoto-meter. Concentration of c h l o r o x y l e n o l and methyl paraben = 5.0 mg l -"'". A b s o r p t i v i t y value of p r e s e r v a t i v e at i s o a b s o r p t i v e wavelength, 232 nm = 28.39. 86 1.7P CM n CM CM CM o 100 CHLOROXYLENOL (%) Fig. 13. Q curve for chloroxylenol and methyl paraben. Points experimental, line fitted using least squares method. Slope = 0.952. Intercept = 0.556. Correlation coefficient = 0.999. 87 A Cx = — - Cy (Eq.52) a4 where: Q q = Absorbancy r a t i o of p r e s e r v a t i v e mixture (X + Y ) . Qx = Absorbancy r a t i o of p r e s e r v a t i v e X. Qy = Absorbancy r a t i o of p r e s e r v a t i v e Y. Absorbancy r a t i o = (absorbance a t ^ max cof. p r e s e r v a t i v e Y)/(absorbance at i s o a b s o r p t i v e p o i n t ) . Fy = F r a c t i o n of p r e s e r v a t i v e 'Y1 i n a p r e s e r v a t i v e mixture (X + Y ) . Cx = Concentration of p r e s e r v a t i v e X. Cy = Concentration of p r e s e r v a t i v e Y. A^ = Absorbance at i s o a b s o r p t i v e wavelength. a^ = A b s o r p t i v i t y value of p r e s e r v a t i v e X or Y at i s o a b s o r p t i v e wavelength. G. P e r m e a b i l i t y of Cellophane Membranes to Nonionic Surfactants (a) E q u i l i b r i u m d i a l y s i s D i a l y s i s c e l l s s i m i l a r to those described by P a t e l and Foss (1964) were used. The two chambers of the c e l l s were separated by cellophane or s i l i c o n e rubber membranes. Twenty m i l l i l i t e r s of a s u r f a c t a n t s o l u t i o n were pipeted i n t o one chamber and 20 ml of d i s t i l l e d water was pipeted i n t o the other. The c e l l s were tumbled i n a temperature-controlled water bath. When the s u r f a c t a n t was.cetomacrogol, equal volumes of the s o l u t i o n s were pipeted at 12-hour i n t e r v a l s from both chambers of the d i a l y s i s c e l l and analyzed f o r s u r f a c t a n t c o n c e n t r a t i o n . For Texofor s u r f a c t a n t s (A„_, A, r, A,_), the 30' 45' 60 s o l u t i o n of the aqueous compartment of the d i a l y s i s c e l l was analyzed q u a l i -t a t i v e l y using the phosphomolybdic a c i d a t e s t f o r the presence of nonionic 88 s u r f a c t a n t at the end of the f o u r t h day. (b) Dynamic d i a l y s i s Twenty!five m i l l i l i t e r s of 10% cetomacrogol was t r a n s f e r r e d to a F i s h e r cellophane bag suspended i n 200 ml of d i s t i l l e d water i n a jacketed beaker. The s o l u t i o n i n the jacketed beaker was s t i r r e d w i t h a magnetic s t i r r e r , w h i l e the cetomacrogol s o l u t i o n i n the cellophane bag was s t i r r e d w i t h a g l a s s s t i r r e r . One hundred m i l l i l i t e r s of the s o l u t i o n was pipeted from the jac k e t e d beaker at 12-hour i n t e r v a l s and analyzed f o r cetomacrogol. The volume of s o l u t i o n i n the jacketed beaker was immediately made up to 200 ml w i t h f r e s h d i s t i l l e d water, so as to maintain s i n k c o n d i t i o n s . (c) U l t r a f i l t r a t i o n A V i s k i n g cellophane membrane was cut and f i t t e d i n t o an Amicon u l t r a - f i l t r a t i o n cell.« T h i r t y f i v e m i l l i l i t e r s of 1.8% cetomacrogol s o l u t i o n was placed i n the c e l l and a pressure of 40 l b / s q i n c h was a p p l i e d u n t i l complete f i l t r a t i o n of the l i q u i d was a t t a i n e d . The f i l t r a t e was analyzed f o r cetomacrogol. H. Binding of C h l o r o c r e s o l w i t h S i l i c o n e Rubber i n E q u i l i b r i u m D i a l y s i s The s i l i c o n e membrane was b o i l e d i n d i s t i l l e d water and subsequently washed w i t h s e v e r a l changes of d i s t i l l e d water. The membrane was placed between the compartments of the d i a l y s i s c e l l . Twenty m i l l i l i t e r s of c h l o r o -c r e s o l s o l u t i o n (of v a r y i n g concentrations) was pipeted i n t o one compartment and 20 ml of water was pipeted i n t o the other. The c e l l s were tumbled u n t i l the concentrations of c h l o r o c r e s o l i n both compartments were the same (four days). The c h l o r o c r e s o l i n both chambers was analyzed s p e c t r o p h o t o m e t r i c a l l y 89 at 280 nm. and the percentage recovery was c a l c u l a t e d to estimate membrane bi n d i n g . I . I n t e r a c t i o n of P r e s e r v a t i v e s w i t h Nonionic Surfactants (a) E q u i l i b r i u m d i a l y s i s technique Two chambered d i a l y s i s c e l l s s i m i l a r to those described by P a t e l and Foss (1964) were used. S i l i c o n e rubber (phenols and parabens), nylon (benzoic a c i d and s o r b i c acid) and cellophane ( c h l o r o c r e s o l ) were used as d i a l y s i s membranes. An aqueous s o l u t i o n of p r e s e r v a t i v e i n the s u r f a c t a n t was placed i n one s i d e of the c e l l and water or water plus p r e s e r v a t i v e was placed i n the other. For the benzoic a c i d and s o r b i c a c i d study, 0.01N HCl, i n s t e a d of water, was used as the s o l v e n t . Two g l a s s beads were added to each compartment to ensure continuous mixing. The c e l l s were r o t a t e d i n a water bath maintained at constant temperature. The p r e s e r v a t i v e i n both chambers was analyzed s p e c t r o p h o t o m e t r i c a l l y at e q u i l i b r i u m (four days f o r c h l o r o -c r e s o l and 7 days f o r parabens, benzoic a c i d and s o r b i c a c i d ) . For the i n t e r -a c t i o n of c h l o r o c r e s o l w i t h nonionic s u r f a c t a n t s , the volume of s o l u t i o n i n each chamber of the d i a l y s i s c e l l was measured at the end of the f o u r t h day. (b) The d i a f i l t r a t i o n technique (I) The d i a f i l t r a t i o n apparatus; The apparatus used i n ' t h i s study i s described w i t h reference to F i g . 3. The Amicon ,r e s e r v o i r tank (12 l i t e r c a p a c i t y ) , w i t h a maximum pressure c a p a c i t y of 100 p s i , i s made of s t a i n l e s s s t e e l (epoxy coated) and f i t t e d w i t h a f i l l - p o r t pressure r e l i e f v a l v e , and i n l e t and output 90 connectors. The r e s e r v o i r was kept i n a temperature c o n t r o l l e d water bath. The Amicon U l t r a f i l t r a t i o n C e l l (Model 52, 50 ml c a p a c i t y , 43 mm diameter membrane) was seated i n a water-jacketed beaker maintained at constant temperature, and the contents s t i r r e d by the use of a magnetic s t i r r e r . D e t a i l s of c e l l s t r u c t u r e and m a t e r i a l s of c o n s t r u c t i o n are given elsewhere (Amicon P u b l i c a t i o n No. 403 A). The u l t r a f i l t r a t i o n membrane used i n t h i s study was UM05. This membrane i s n e g a t i v e l y charged and i s s a i d to prevent the passage of molecules of molecular weight greater than 600. An Amicon C/D ( c o n c e n t r a t i o n / d i a l y s i s ) S e l e c t o r was connected to a n i t r o g e n c y l i n d e r , r e s e r v o i r , and the u l t r a f i l t r a t i o n c e l l as shown i n F i g . 3. This allowed gas or l i q u i d to flow from the r e s e r v o i r to the c e l l as de s i r e d and thus the s o l u t i o n i n the c e l l was concentrated or d i a l y z e d r e s p e c t i v e l y . ( I I ) Procedure f o r the determination of benzoic acid-cetbmacrogol  i n t e r a c t i o n using the d i a f i l t r a t i o n technique: (1) The u l t r a f i l t r a t i o n c e l l was charged w i t h cetomacrogol s o l u t i o n . (2) The r e s e r v o i r was f i l l e d w i t h benzoic a c i d s o l u t i o n i n 0.01N HCl at the maximum conc e n t r a t i o n of i n t e r e s t . (3) An Amicon C/D S e l e c t o r , w i t h the v a l v e i n the 'GAS' p o s i t i o n , was connected w i t h the r e s e r v o i r , u l t r a f i l t r a t i o n c e l l and the pressure source ( N 2 c y l i n d e r ) , thus p e r m i t t i n g gas to pass from the r e s e r v o i r tank to the c e l l . With the pressure r e l i e f v a l v e s on the tank and the c e l l both i n the open p o s i t i o n , the pressure v a l v e of the n i t r o g e n c y l i n d e r was opened s l o w l y . A f t e r c l o s i n g both pressure v a l v e s , the c y l i n d e r pressure v a l v e was opened 91 u n t i l the desired pressure was achieved on the cylinder gauge. A time of two minutes was allowed.for the pressure to equalize i n the tank and the c e l l . The C/D se l e c t o r switch was s h i f t e d to the 'LIQUID' p o s i t i o n , and the pressure r e l i e f valve on the c e l l was s l i g h t l y opened to allow the so l u t i o n to begin to flow into the tubing connected to the c e l l . Extreme care was taken i n opening the pressure r e l i e f valve, because i f the valve was opened too much, s o l u t i o n flow was excessive, and a volume increase occurred i n the c e l l . (4) The u l t r a f i l t r a t e was c o l l e c t e d automatically by a n l s c o F r a c t i o n C o l l e c t o r . Samples were c o l l e c t e d on a volume bas i s . This procedure involved the use of a volume c o l l e c t i n g device but checks on volumes delivered indicated that the device tended to be inaccurate. Hence, a l l f r a c t i o n s c o l l e c t e d were checked independently f o r volume. (5) The concentration of benzoic acid i n each successive f r a c t i o n , [D^], was determined spectrophotometrically at 230 nm. (6) The concentration of preservative bound to the surfactant, [D^], foraa given free preservative concentration i n the u l t r a f i l t r a t e , [D^], was calculated using the following equations (see appendix 1): (Eq.53) (Eq.54) 1000 (Eq.55) where: A = Amount of preservative entering the c e l l . = Amount of preservative bound to surfactant. 92 = Amount of preservative i n a f r a c t i o n of u l t r a f i l t r a t e . A^ = Amount of preservative free i n the c e l l . L-D ] = Concentration of preservative i n the r e s e r v o i r . [D^] = Concentration of preservative bound to surfactant. [D^] = Concentration of free preservative. V = Volume of the surfactant s o l u t i o n i n the c e l l , m V^ = Volume of a f r a c t i o n of u l t r a f i l t r a t e . (7) The f i n a l volume of the cetomacrogol s o l u t i o n was recorded at the end of the experiment. J. Interaction of Preservative Mixtures with Cetomacrogol The equilibrium d i a l y s i s technique was used to study the i n t e r -action of preservative mixtures with cetomacrogol. The d i a l y s i s membrane was e i t h e r nylon (benzoic acid and sorbic acid mixture) or s i l i c o n e rubber (phenols and parabens mixtures). Twenty m i l l i l i t e r s of aqueous cetomacrogol so l u t i o n plus preservative (D) or competitor (C) or both was placed i n one side of the two-chambered d i a l y s i s c e l l and 20 ml of water or water plus preservative (D) or competitor (C) or both was placed i n the other. For the benzoic acid and sorbic acid study, 0.01N HCl, instead of water, was used as the solvent. The experiments were so designed that i n each study the concentration of preservative (D) varied while the concentration of the competitor (C) was kept constant. The time required to reach equilibrium ranged from four to seven days. The concentration of preservative (D) and competitor (C) i n both chambers was analyzed spectrophotometrically using the absorbancy-ratio method. 93 K. Interaction of Chlorocresol with Mixtures of Nonionic Surfactants Methodology similar to that used for the interaction of preser-vatives with nonionic surfactants using equilibrium dialysis was ut i l i z e d to study interaction of chlorocresol with surfactant mixtures. The dialysis membrane was silicone rubber. The surfactant solution in the dialysis c e l l was replaced with an aqueous solution of surfactant mixture. L. Distribution of Chlorocresol in Liquid Paraffin-Water Systems Equal volumes of aqueous chlorocresol solution and liquid paraffin were pipeted into glass-stoppered flasks and agitated using a wrist-action shaker for about one hour at room temperature. The flasks were transferred to a temperature controlled shaker bath and agitated t i l l equilibrium was reached (three days). The aqueous phase was separated by centrifugation of the oil-water mixture and analyzed spectrophotometrically for chlorocresol concentration. The concentration of chlorocresol in the o i l phase was calculated by subtracting the amount of chlorocresol in the aqueous phase from the total amount of chlorocresol added. M. Distribution of Chlorocresol in Liquid Paraffin-Water-Cetomacrogol. Systems Liquid paraffin and cetomacrogol solutions were mixed in various ratios and passed through hand powered homogenizers, at least five times, to ensure the formation of stable emulsions. For microbiological work the emulsions were prepared aseptically in 1 a.laminar flow hood using sterile liquid paraffin and cetomacrogol solutions. The homogenizers were sterilized by autoclaving at 121° for 30 minutes. 94 For emulsions c o n t a i n i n g c h l o r o c r e s o l , p r i o r to homogenization, the l i q u i d p a r a f f i n and cetomacrogol s o l u t i o n s c o n t a i n i n g c h l o r o c r e s o l were mixed and a g i t a t e d i n a water bath maintained at a constant temperature f o r about 3 days, a time s u f f i c i e n t f o r the p r e s e r v a t i v e to e q u i l i b r a t e between va r i o u s phases of the emulsion. An emulsion c o n t a i n i n g v a r i o u s amounts of c h l o r o c r e s o l was pipeted i n t o one compartment of a d i a l y s i s c e l l f i t t e d w i t h a s i l i c o n e rubber membrane. An aqueous s o l u t i o n c o n t a i n i n g v a r i o u s amounts of c h l o r o c r e s o l was pipeted i n t o the other compartment. The c e l l s were e q u i l i b r a t e d (about 4 days) and the conc e n t r a t i o n of c h l o r o c r e s o l i n the aqueous compartment was analyzed s p e c t r o p h o t o m e t r i c a l l y at 225 nm. N. M i c r o b i o l o g i c a l Procedures (a) Organism: R e p l i c a t e s l a n t s of E. c o l i (ATCC 8739) were used i n a l l the experiments. The s l a n t s had been prepared from a s i n g l e colony and were stored i n , a r e f r i g e r a t o r at 4°. (b) C u l t u r e media: T r y p t i c a s e soy br o t h and t r y p t i c a s e soy agar were rehydrated and s t e r i l i z e d according to the s p e c i f i c a t i o n s of the manufacturer. Deionized g l a s s d i s t i l l e d water, 0.85% s a l i n e and peptone water .(each 1500 ml) were s t e r i l i z e d by a u t o c l a v i n g at 121° f o r 35 minutes. (c) S t e r i l i z a t i o n of experimental s o l u t i o n s : Aqueous c h l o r o c r e s o l s o l u t i o n s and cetomacrogol s o l u t i o n s w i t h and without c h l o r o c r e s o l were s t e r i l i z e d by passing the s o l u t i o n through s t e r i l e M i l l i p o r e 0.45/*HA f i l t e r s . L i g h t l i q u i d p a r a f f i n was s t e r i l i z e d by dry heat at 160° f o r one hour. 95 (d) S t e r i l i z a t i o n of M i l l i p o r e f i l t r a t i o n equipment: A M i l l i p o r e 6 place s t e r i l i t y test manifold u n i t , M i l l i p o r e f f u n n e l s and M i l l i p o r e 0.45/»HAEG f i l t e r s were s t e r i l i z e d according to s p e c i f i c a t i o n s of the manufacturer ( M i l l i p o r e Catalogue No. MRP-4). . (e^ B a c t e r i a l cultures: An aliquot (5 ml) of try p t i c a s e soy broth was inoculated from a fresh s l a n t , and the culture was allowed to grow for 12 hours at 37° i n an incubator. A sample of 0.2 ml of t h i s culture was transferred to 50 ml of fresh t r y p t i c a s e soy broth and incubated at 37° for 12 hours. This f i n a l E. c o l i suspension was used for the construction of a standard curve and for the preparation of standard E_. c o l i suspensions for experimental work. The p u r i t y of the culture was checked using Gram's sta i n i n g procedure and examining the growth on MacConkey agar. (f) Construction of standard curve for E. c o l i : A series of d i l u t i o n s of —• c o 1 1 suspension were made i n try p t i c a s e soy broth and the. absorbance measured at 550 nm. using a double beam spectrophotometer. Trypticase soy broth was used as blank i n the absorbance measurements. An aliqu o t from each d i l u t i o n was appropriately d i l u t e d with 0.85% s a l i n e and v i a b l e counts were made using the pour plate technique. (g) Preparation of standard E. c o l i suspension: An aliqu o t of the culture was d i l u t e d with t r y p t i c a s e soy brothaand the absorbance measured at 550 nm. From the absorbance, the d i l u t i o n - f a c t o r and with reference to the.standard curve (Fig. 14), the concentration of E. c o l i i n the culture'was determined. The culture was d i l u t e d with 0.85% s a l i n e to produce the desired number of E. c o l i per ml of suspension. 0.7 100 200 300 Number of E. c o l i x 10 /ml 97 (h) Viable count method using pour-plate technique: Samples of 0.5 ml were withdrawn from the cultures and appropriately d i l u t e d into s t e r i l i z e d 0.85% s a l i n e so that 50-200 colonies per plate would r e s u l t . From these d i l u t i o n s , a l i q u o t s of 1 ml were pipeted into each of three p e t r i p l a t e s and to each plate 12 ml of s t e r i l e t r y p t i c a s e soy agar, kept at 45° i n water bath, was poured. The plates were rotated to disperse the culture i n agar and incubated f o r 48 hours at 37°. The colonies were counted using a colony counter. . (i) Viable count method using membrane f i l t r a t i o n technique: A M i l l i p o r e 6 place S t e r i l i t y Test Manifold unit with 47 mm edge-hydrophobic M i l l i p o r e f i l t e r s (HAEG, 0.45/?) was used. D e t a i l s of the M i l l i p o r e f i l t r a t i o n assembly setup and operation are given elsewhere ( M i l l i p o r e Catalogue No. MRP-4). A l l operations were performed under a laminar flow hood. Samples of 0.5 ml were appropriately d i l u t e d with s t e r i l e normal s a l i n e to produce 100 to 200 colonies per M i l l i p o r e f i l t e r . From these d i l u t i o n s aliquots of 1 ml were pipeted into M i l l i p o r e funnels containing approximately 10 ml of peptone water and vacuum was applied immediately to draw the sample through 'the f i l t e r . The f i l t e r was washed 5 times with 25 ml portions of peptone water, and each time vacuum was applied immediately. Upon completion of f i l t r a t i o n , the f i l t e r was transferred to tr y p t i c a s e soy agar p l a t e s , and incubated at 37° for 24 hours. The colonies were counted using a colony counter. For chlorocresol treated E_. c o l i the plates were incubated f o r another 12 hours, and colonies recounted to check f or the emergence of new colonies. 98 ( j ) B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n water: Aqueous c h l o r o c r e s o l s o l u t i o n s (41.5 ml) were i n o c u l a t e d w i t h 0.5 ml of standard E_. c o l i suspen-s i o n so that each ml of s o l u t i o n contained 10 organisms. Decrease i n p r e s e r v a t i v e c o n c e n t r a t i o n due to the a d d i t i o n of 0.5 ml of c u l t u r e was taken i n t o account i n the c a l c u l a t i o n of i n i t i a l c h l o r o c r e s o l concentrations. The c h l o r o c r e s o l s o l u t i o n s were a g i t a t e d i n a water bath maintained at 25°, samples were withdrawn at given time i n t e r v a l s and v i a b l e counts made using the membrane f i l t r a t i o n technique. Controls c o n s i s t i n g of d i s t i l l e d water were s i m i l a r l y run w i t h each experiment to check the v i a b i l i t y of the organisms during the experimental p e r i o d . (k) B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n cetomacrogol s o l u t i o n s : Ceto-macrogol s o l u t i o n s of v a r y i n g c o n c e n t r a t i o n were made i n d i s t i l l e d water. The t o t a l c o n c e n t r a t i o n of c h l o r o c r e s o l , [D ] , g i v i n g the d e s i r e d f r e e p r e s e r v a t i v e c o n c e n t r a t i o n i n the aqueous phase [D^], was. c a l c u l a t e d using Eq.28. A l l cetomacrogol s o l u t i o n s had the same [D f] but d i f f e r e n t [D ] . Cor r e c t i o n s were made i n the i n i t i a l cetomacrogol and c h l o r o c r e s o l concentra-t i o n s to account f o r the d i l u t i o n of f i n a l s u r f a c t a n t s o l u t i o n due to the a d d i t i o n of 0.5 ml of E. c o l i suspension. Cetomacrogol s o l u t i o n s (41.5 ml) co n t a i n i n g c h l o r o c r e s o l were a g i t a t e d i n a water bath f o r about 3 hours to e q u i l i b r a t e the p r e s e r v a t i v e between the aqueous and m i c e l l a r phases of the s u r f a c t a n t s o l u t i o n at given temperature. At e q u i l i b r i u m , the cetomacrogol s o l u t i o n s were i n o c u l a t e d w i t h 0.5 ml of standard jE. c o l i c u l t u r e so that each ml of s u r f a c t a n t s o l u t i o n contained 10^ organisms. The cetomacrogol s o l u t i o n s were sampled at given time i n t e r v a l s and v i a b l e counts were made using the membrane 99 f i l t r a t i o n technique. Controls consisting of aqueous cetomacrogol solutions were run with each experiment to check the v i a b i l i t y of the organisms during the experimental period. (1) Bactericidal activity of chlorocresol in liquid-paraffin emulsions stabilized with cetomacrogol: Liquid paraffin emulsions of varying oil-water ratios were prepared aseptically (see method for the preparation of emulsions) using st e r i l e liquid paraffin and cetomacrogol solutions. The total chlorocresol concentration, [D], required in an emulsion to get the desired free preservative concentration in the aqueous phase, [D^], was calculated using Eq.48. Corrections were made in the i n i t i a l cetomacrogol and chlorocresol concentrations to account for the dilution of the f i n a l emulsion due to the addition of 0.5. ml of E. c o l i suspension.. Freshly homogenized liquid paraffin emulsions (41.5 ml) containing chlorocresol were agitated in a water bath for about 3 hours to equilibrate the preservative between the various phases of the emulsion. The emulsions were inoculated with 0.5 ml of standard El. c o l i culture so that each ml of 6 emulsion contained 10 organisms. The emulsions were sampled at given time intervals and viable counts were made using the membrane f i l t r a t i o n technique. Controls consisting of blank liquid paraffin emulsions were similarly run with each experiment to check the v i a b i l i t y of the organisms during the experimental period. 100 RESULTS AND DISCUSSION A. P e r m e a b i l i t y of Membranes to Nonionic Surfactants (a) . P e r m e a b i l i t y of membranes to Texofor s u r f a c t a n t s Q u a l i t a t i v e t e s t s of the s o l u t i o n of the aqueous compartment of the d i a l y s i s c e l l showed that the F i s h e r cellophane membrane was permeable to Texofors (cetomacrogol), A ^ Q , A^ <. and A ^ Q . The s i l i c o n e rubber was t o t a l l y impermeable to the same s u r f a c t a n t s . (b) P e r m e a b i l i t y of membranes to cetomacrogol Figure 15 shows the f r a c t i o n of cetomacrogol d i a l y z e d through F i s h e r and V i s k i n g cellophane membranes as a f u n c t i o n of time i n e q u i l i b r i u m d i a l y s i s . Both membranes were permeable to cetomacrogol at approximately the same r a t e . Figure 16 shows the p e r m e a b i l i t y of F i s h e r cellophane membrane to cetomacrogol using dynamic d i a l y s i s under s i n k c o n d i t i o n s . The r a t e of permeation decreases w i t h increase i n time. This i s p o s s i b l y due to r a p i d permeation of low molecular weight f r a c t i o n s , followed by a slow d i f f u s i o n of the molecules of higher molecular weight. U l t r a f i l t r a t i o n of cetomacrogol s o l u t i o n (1.8%) through V i s k i n g cellophane membrane showed that 2.7% of the t o t a l s u r f a c t a n t passed through the membrane. B. Volume Change due to Osmosis i n D i a l y s i s Studies Increase i n the volume of s o l u t i o n i n the s u r f a c t a n t chamber of 101 HOURS Permeab.ility of cellophane membranes to cetomacrogol in equilibrium dialysis. I n i t i a l cetomacrogol concentrations: • , 38.46 x 10~3; O » 76.92 x 10"3 moles l - 1 . Open and closed symbols represent Fisher and Visking cellophane membranes respectively. 102 HOURS P e r m e a b i l i t y of F i s h e r cellophane membrane to cetomacrogol i n dynamic d i a l y s i s under s i n k c o n d i t i o n s . I n i t i a l cetomacrogol concentration was 76.92 x 10 moles 1~1. The f r a c t i o n d i a l y z e d was not correct e d f o r changes i n s u r f a c t a n t c o n c e n t r a t i o n due to osmosis. 103 the equilibrium d i a l y s i s c e l l and the cellophane bag used i n dynamic d i a l y s i s technique.showed that osmosis had occurred. Table 3 shows the volume changes i n an equilibrium d i a l y s i s study of the i n t e r a c t i o n of chl o r o c r e s o l with some Texofor surfactants when Fisher cellophane was used as the semipermeable membrane. F i f t e e n to f o r t y percent of the water was removed from the aqueous compartment of the d i a l y s i s c e l l , thus causing appreciable d i l u t i o n of the so l u t i o n i n the surfactant compart-ment. C. Interaction of Preservatives with Nonionic Surfactants (a) Equilibrium d i a l y s i s technique (i) Interaction of chl o r o c r e s o l with Texofor surfactants: Table 4 shows a comparison between the binding r e s u l t s obtained using s i l i c o n e rubber and Fish e r cellophane membranes i n equilibrium d i a l y s i s studies i n v o l v i n g the i n t e r a c t i o n of chlorocresol with Texofor A^Q, A ^ and A^Q. For a given i n i t i a l surfactant concentration the t o t a l amount of chlorocresol i n the d i a l y s i s c e l l was the same for both s i l i c o n e rubber and cellophane membranes. However, the values of r obtained using cellophane membranes are lower than those obtained using s i l i c o n e rubber membranes. Since, the c e l l o -phane membrane was permeable to the surfactants, and osmosis occurred during d i a l y s i s (Table 3 ) , the observed decrease i n the values of r can be ascribed both to osmosis and surfactant permeation through the membrane. This aspect w i l l be discussed further i n the following section. C i t ) Interaction of chlorocresol with cetomacrogol: The binding r e s u l t s i n the form of a Scatchard p l o t (Scatchard, 1949) are shown i n 104 TABLE 3. Volume change due to osmosis i n e q u i l i b r i u m d i a l y s i s using F i s h e r cellophane as a semipermeable membrane. Texofor I n i t i a l S u rfactant Concentration % Volume i n Surfactant Compartment ml Volume i n Aqueous Compartment ml A 3 0 5.0 10.0 22.8 25.2 16.6 ' , Li > 14.2 A 4 5 5.0 10.0 24.1 26.5 15.1 12.5 A 6 0 5.0 10.0 24 27.4 15.2 11.9 1. I n i t i a l volumes i n the aqueous and s u r f a c t a n t compartments were 20 ml. 2. Volumes were measured at the end of the 4th day. 3. Volumes were determined using p i p e t s , t h e r e f o r e , complete recovery of f l u i d s was not p o s s i b l e . Thus, the volumes shown i n the t a b l e represent approximate v a l u e s . TABLE 4. Comparison of " r " f o r a given t o t a l amount of c h l o r o c r e s o l i n e q u i l i b r i u m d i a l y s i s using s i l i c o n e rubber and F i s h e r c e l l o -phane as semipermeable membranes. Texofor I n i t i a l S urfactant r = [D b]/[M]* Concentration % S i l i c o n e Membrane F i s h e r C e l l o -phane Membrane 5.0 1.68 1.47 A 3 0 10.0 • i 7* 1.75 1.37 5.0 1.173 " 1.37 A 4 5 : 10.0 1.89 1.38 550 . 1.76 1.43 A60 10.0 1.98 1.39 * The s u r f a c t a n t c o n c e n t r a t i o n , [ M ] , was not co r r e c t e d f o r d i l u t i o n due to osmosis, or l o s s due to permeation through the d i a l y s i s membrane. 106 Figure 17. The binding of chlorocresol with cetomacrogol i s independent of surfactant concentration when s i l i c o n e rubber i s used as the d i a l y s i s membrane. Similar independence of surfactant concentration has been reported previously for the binding of several other preservatives with cetomacrogol when nylon was used as the d i a l y s i s membrane (Kazmi and M i t c h e l l , 1970). The curve i n F i g . 17 was characterized according to Eq.25 on the assumption that two classes of binding s i t e s are.involved i n the i n t e r a c t i o n . [D,] n l K l [ D f ] n 2 K 2 [ D £ ] = r = + (Eq.25) M l+K 1[D f] l+K 2[D f] The experimental data are indicated by points while the s o l i d l i n e s were f i t t e d according to the n and K values computed from Eq.25 using a non-linear regression program (see appendix 2). Figure 17 shows that when cellophane d i a l y s i s membrane was used, the binding i s apparently not independent of cetomacrogol concentration and a s e r i e s of curves are obtained. T h e o r e t i c a l l y such r e s u l t s i n d i c a t e that an increase i n the concentration of cetomacrogol r e s u l t s i n a decrease i n the number of binding s i t e s on the cetomacrogol molecule. However, since the r e s u l t s obtained with s i l i c o n e rubber membrane are independent of surfactant concentration, i t i s suggested that changes i n cetomacrogol concentration due to d i l u t i o n as a r e s u l t of osmosis and/or to permeation of surfactant through the cellophane membrane provide a more l i k e l y explanation. Permeation of s u f f i c i e n t cetomacrogol into the "surfactant-f r e e " chamber of the d i a l y s i s c e l l would lead to an increase i n the apparent value of [D £] due to m i c e l l a r i n t e r a c t i o n with the c h l o r o c r e s o l and, therefore, 107 Fig. 17. Scatchard plot for the interaction of chloro-cresol with cetomacrogol. I n i t i a l cetomacrogol concentrations: O , 7.69 x 10~3; 7^, 15.3 x 10 - 3 • , 23.07 x 10~3; Q , 38.46 x 10 - 3; & , 76.92 x 10" 3 moles l ~ x . Open and closed symbols represent data obtained using silicone and Fisher cellophane membranes respectively. 108 decrease the r / [ D £ ] r a t i o f o r a given r value . -Moreover, l o s s of s u r f a c t a n t by permeation through the membrane would a l s o decrease r due to the un-cor r e c t e d decrease i n the value of [M] and the decrease i n [D^]. Thus, a p l o t of r/£D£J versus r f o r d i f f e r e n t concentrations of cetomacrogol would r e s u l t i n a s e r i e s of curves each representing a given s u r f a c t a n t concen-t r a t i o n . D i l u t i o n of cetomacrogol as a r e s u l t of osmosis would produce a s i m i l a r displacement of the bi n d i n g curves. The s u r f a c t a n t c o n c e n t r a t i o n r e q u i r e d to produce the observed displacement of the b i n d i n g curves was c a l c u l a t e d from a rearrangement of Eq.25. 1 [D b] (1 + K ^ ] + K 2 [ D f ] + K lK 2 [Dg ] 2 ) [MJ = : : (Eq.56) [D f] ( n l K l + n 2 K 2 + n ^ K ^ ] + n ^ K ^ ^ ] ) where D^] and [D £] are the experimental values f o r cellophane membrane ( F i g . 17) and n^, K^, n 2 , K 2 are" the b i n d i n g constants obtained using s i l i c o n e rubber membrane. The data i n Table 5 shows that the changes i n s u r f a c t a n t c o n c e n t r a t i o n ( i . e . , the d i f f e r e n c e s between the i n i t i a l and c a l c u l a t e d cetomacrogol c o n c e n t r a t i o n s , M^ - M^) were co n s i d e r a b l y greater than could be accounted f o r s o l e l y by l o s s of s u r f a c t a n t as a r e s u l t of permeation through the cellophane membrane, M^. However, when the bi n d i n g curves are r e p l o t t e d using s u r f a c t a n t concentrations c o r r e c t e d f o r volume changes i n the chambers of the d i a l y s i s c e l l , M 2 > the d i s c r e p a n c i e s between the curves determined using s i l i c o n e rubber membrane and cellophane membrane i s markedly reduced ( F i g . 18). Since the d i f f e r e n c e between M 2 and M^ i s of the same order of 109 TABLE 5. Change of Surfactant Concentration in Equilibrium Dialysis with Cellophane as a Semipermeable Membrane -1 3 Cetomacrogol Concentration Moles 1,- x 10 Mx M2 M3 M4 I n i t i a l Calculated f rou Calculated Permeated at from Volume using Eq.56^ 96 hr c Change3 38.46 33.88 31.60 2.19 76.92 62.06' 55.42 7.23 (a) Each value represents the mean of five readings. (b) Values were obtained by substituting n^ K^ and i^K^ calculated from the binding curve obtained using silicone rubber membrane (Fig. 17), and [D^] and [D^], obtained using cellophane membrane (Fig. 17), into Eq.56. Each value represents the mean of five readings.?' I (c) From Figure 15. 110 11 10 CNJ I o 4-4 1 ! i-l 0.4 0.8 1.2 1.6 2.0 F i g . 18. Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol. Upper b i n d i n g curve obtained using s i l i c o n e rubber membrane as i n F i g . 17 . Cellophane membrane data corrected f o r changes i n s u r f a c t a n t concentrations due to osmosis; i n i t i a l concentrations as i n F i g . 17;. co r r e c t e d concentrations shown i n Table 5. I l l magnitude as M^, the r e s i d u a l displacement of the bi n d i n g curves can be a t t r i b u t e d to the decrease i n [M] due to p e r m e a b i l i t y of the cellophane membrane to cetomacrogol. Hence, i t can be concluded that the observed displacement of the bi n d i n g curves obtained i n the e q u i l i b r i u m d i a l y s i s study u s i n g cellophane membranes i s due both to osmosis and to p e r m e a b i l i t y of the membrane to the s u r f a c t a n t , w i t h d i l u t i o n of s u r f a c t a n t as a r e s u l t of the osmotic d i f f e r -e n t i a l across the membrane being the major f a c t o r . I t i s apparent that the use of cellophane as a membrane w i l l introduce appreciable e r r o r s i n t o i n t e r a c t i o n and tr a n s p o r t s t u d i e s i n v o l v i n g nonionic s u r f a c t a n t s unless c o r r e c t i o n s are made f o r osmosis of the experimental design i s such that osmosis i s avoided. (b) D i a f i l t r a t i o n technique F i g u r e 19 i s a Scatchard p l o t (Scatchard, 1949) f o r the i n t e r -a c t i o n of benzoic a c i d w i t h cetomacrogol. The b i n d i n g curve d e r i v e d from the e q u i l i b r i u m d i a l y s i s data i s independent of cetomacrogol c o n c e n t r a t i o n . This i s i n agreement w i t h the benzoic acid-cetomacrogol i n t e r a c t i o n s reported i n the l i t e r a t u r e ( M i t c h e l l and Brown, 1966; Donbrow, Azaz and Hamburger, 1970; Kazmi and M i t c h e l l , 1971). However, a p l o t of d i a f i l t r a t i o n data give two separate b i n d i n g curves f o r 1% and 5% cetomacrogol concentrations r e s p e c t -i v e l y . An increase i n cetomacrogol c o n c e n t r a t i o n r e s u l t s i n a downward s h i f t of the b i n d i n g curve. For a given cetomacrogol c o n c e n t r a t i o n the b i n d i n g curve i s reasonably r e p r o d u c i b l e . Thus the d i a f i l t r a t i o n r e s u l t s i n d i c a t e that the simple Law of Mass A c t i o n does not adequately describe the benzoic acid-cetomacrogol i n t e r a c t i o n , and the bindi n g i s not independent of 112 12 10 o X 1—I 4-Q I I r x 10 F i g . 19. Scatchard p l o t f o r the i n t e r a c t i o n of benzoic a c i d with. c e t o ~ macrogol. Cetomacrogol concentrations: 0\ 7.69 x 10^ 3,O , 38.46 x 10 moles l - x . Open and c l o s e d symbols represent data obtained using e q u i l i b r i u m d i a l y s i s and e q u i l i b r i u m u l t r a f i l t r a t i o n techniques r e s p e c t i v e l y . 113 macromolecule con c e n t r a t i o n . This i s very u n l i k e l y . S i m i l a r dependence of b i n d i n g curves of macromolecule con c e n t r a t i o n have been reported f o r drug-protein i n t e r a c t i o n s u s i n g the d i a f i l t r a t i o n technique ( B l a t t , Robinson and B i x l e r , 1968; Ryan and Hanna, 1971; Crawford, Jones, Thompson and We l l s , 1972; Palmer, 1972; Barnes, M i t c h e l l , Palmer and Pernarowski, 1973). Attempts have been made to e x p l a i n the anomalous b i n d i n g r e s u l t s obtained u s i n g d i a f i l t r a t i o n technique i n drug-protein i n t e r a c t i o n s t u d i e s . B l a t t , Robinson and B i x l e r (1968) explained t h e i r r e s u l t s i n terms of p o l a r -i z a t i o n (or caking) of drug molecules at the membrane. To overcome t h i s problem they suggested using the lowest p o s s i b l e pressures and low macro-molecule concentrations or l a r g e p o r o s i t y membranes. The p o s s i b i l i t y of p r e s e r v a t i v e p o l a r i z a t i o n cannot be r u l e d out i n t h i s study because a high 2 " pressure (9.1 kg/m ) was used to enhance the f l u x r a t e of the aqueous phase through UM05 membrane. U n l i k e drug-protein i n t e r a c t i o n s t u d i e s , the use of la r g e p o r o s i t y membranes to a l l e v i a t e p r e s e r v a t i v e p o l a r i z a t i o n i n p r e s e r v a t i v e - s u r f a c t a n t i n t e r a c t i o n s t u d i e s i s not f e a s i b l e here. P o s s i b l e s u r f a c t a n t permeation through the membrane w i l l introduce appreciable e r r o r s i n t o the b i n d i n g parameters. Even the Amicon UM05 (molecular weight c u t o f f = 500) d i d not hold the cetomacrogol (molecular weight = 1300) completely. Q u a l i t a t i v e t e s t s of the u l t r a f i l t r a t e showed that t r a c e s of s u r f a c t a n t passed through the membrane. The magnitude of the e r r o r s introduced into, the b i n d i n g parameters due to p e r m e a b i l i t y of cetomacrogol through F i s h e r c e l l o -phane membrane i n an e q u i l i b r i u m d i a l y s i s study were pointed out i n the previous s e c t i o n . A s i m i l a r e x p l a n a t i o n can be given f o r the downward s h i f t of the b i n d i n g curves i n the d i a f i l t r a t i o n s t u d i e s of benzoic acid-cetomacrogol i n t e r a c t i o n s . I ncreasing the concentration of cetomacrogol i n the c e l l would r e s u l t i n the permeation of a greater amount of s u r f a c t a n t i n the u l t r a f i l t r a t e , and thus higher value of [D £] due to m i c e l l a r s o l u b i l i z a t i o n of the p r e s e r v a t i v e . This e f f e c t w i l l decrease the r/ [ D £ ] r a t i o , f o r a given r,, and cause a downward s h i f t of the bi n d i n g curve. Drug b i n d i n g and/or r e j e c t i o n by the d i a f i l t r a t i o n apparatus can a l s o a f f e c t the bi n d i n g r e s u l t s ( B l a t t , Robinson and B i x l e r , 1968; Ryan and Hanna, 1971; Palmer, 1972). Attempts to separate the e f f e c t s of drug b i n d i n g and r e j e c t i o n i n d i a f i l t r a t i o n s t u d i e s were unsuccessful ( B l a t t , Robinson and B i x l e r , 1968; Palmer, 1972). The phenomenon of membrane r e j e c t i o n was demonstrated by Ryan and Hanna (1971). U l t r a f i l t r a -t i o n of testosterone s o l u t i o n was carried, out using an Amicon UM05 membrane. Simultaneous sampling of the c e l l content and the u l t r a f i l t r a t e revealed that c e l l t e s t o s t e r o n e l e v e l s rose above the r e s e r v o i r c o n c e n t r a t i o n . The authors a s c r i b e d t h i s observation to the membrane-rejection phenomenon. Palmer (1972) demonstrated the b i n d i n g of bishydroxycoumarin and phenyl-butazone w i t h v a r i o u s p a r t s of the d i a f i l t r a t i o n apparatus. Bishydroxy-coumarin was bound w i t h the d i a f i l t r a t i o n apparatus to a greater extent than phenylbutazone. Binding of drugs and p r e s e r v a t i v e s to p l a s t i c s and s y n t h e t i c membranes i s a common problem ( P a t e l and Kostenbauder, 1958; Kapadia, Guess and A u t i a n , 1964; Ooteghem and Herbots,11969; Schoenwald and B e l c a s t r o , 1969; Chiou and Smith, 1970; Cho, 1970; P a t e l and Nagabhushan, 1970; Flynn and Roseman, 1971; Kakemi et a l . , 1971; Kazmi, 1971; McCarthy, 1972; Nasim et a l . , 1972; Jacob and G i l b e r t , 1973a,b; F i g . 36). The d i a f i l t r a t i o n data f o r the i n t e r a c t i o n of benzoic a c i d w i t h cetomacrogol gave higher values of K than the value of K obtained using e q u i l i b r i u m d i a l y s i s ( F i g . 19). I t i s 115 p o s s i b l e that an increase i n the value of K using d i a f i l t r a t i o n could be due to b i n d i n g of benzoic a c i d w i t h m a t e r i a l s of the d i a f i l t r a t i o n apparatus. I f t h i s i s t r u e , then values of n f o r d i a f i l t r a t i o n b i n d i n g curves should be higher than the values of n obtained u s i n g e q u i l i b r i u m d i a l y s i s t e c h -nique. However, si n c e the reverse i s the case, the higher values of K f o r the d i a f i l t r a t i o n curves must be due to an e f f e c t other than b i n d i n g of benzoic a c i d w i t h d i a f i l t r a t i o n apparatus. Ryan and Hanna (1971) suggested that the membrane p r o p e r t i e s may change during d i a f i l t r a t i o n . A decreased flow r a t e w i t h increase i n p r o t e i n c o n c e n t r a t i o n was reported (Ryan and Hanna, 1971; Palmer, 1972). I t was suggested that the decrease i n the flow r a t e may be due to clogging of the membrane pores w i t h i n c r e a s i n g concentrations of the p r o t e i n . Recently, Palmer e_t a l . (1973) reported f l u x r a t e changes i n the u l t r a f i l t r a t i o n of d i l u t e aqueous s o l u t i o n s of s u r f a c e - a c t i v e and nonsurface-active s o l u t e s through commercially a v a i l a b l e c e l l u l o s e acetate and p o l y s a l t complex (Amicon UM05) membranes. T r i t o n X-100, sodium dodecylbenzenesulfonate, alkyldimethylbenzylammonium c h l o r i d e (Hyamine 3500), and carbowax 600 were chosen as the model nonionic s u r f a c t a n t , a n i o n i c s u r f a c t a n t , c a t i o n i c s u r f a c t a n t , and nonionic symmetrical s o l u t e r e s p e c t i v e l y . F l u x d e c l i n e s r e s u l t i n g from s p e c i f i c solute-membrane i n t e r a c t i o n s were observed f o r T r i t o n X-100 w i t h c e l l u l o s e acetate membranes and f o r the i o n i c s u r f a c e -a c t i v e agents w i t h p o l y s a l t membranes. Although small f l u x d e c l i n e s were observed f o r the i o n i c s u r f a c t a n t s w i t h c e l l u l o s e acetate membranes and f o r the n o n i o n i c s u r f a c t a n t w i t h p o l y s a l t complex membranes, these small f l u x -l i m i t i n g e f f e c t s were w i t h i n the range of those observed f o r the nonsurface-a c t i v e carbowax 600. I n t e r a c t i o n of cetomacrogol and T r i t o n X-100 w i t h 116 M i l l i p o r e VS membrane was demonstrated using e q u i l i b r i u m d i a l y s i s technique (Kazmi, 1971; Kazmi, unpublished r e s u l t s ) . In the present study f l u x r a t e changes due to p o s s i b l e s p e c i f i c or n o n - s p e c i f i c i n t e r a c t i o n of s u r f a c t a n t w i t h the membrane should have l i t t l e e f f e c t on the benzoic acid-cetomacrogol b i n d i n g r e s u l t s because samples of u l t r a f i l t r a t e were c o l l e c t e d on a volume ra t h e r than time b a s i s . Ryan and Hanna (1971) considered the p o s s i b i l i t y of conformational changes i n the bovine serum albumin (BSA) during d i a f i l t r a t i o n . The albumin s o l u t i o n was washed i n the d i a f i l t r a t i o n c e l l f o r 12 hours w i t h b u f f e r . A subsequent e q u i l i b r i u m d i a l y s i s experiment i n d i c a t e d that the washed BSA had a decreased a f f i n i t y f o r t e s t o s t e r o n e . Palmer (1972) suggested that a l t e r a t i o n i n the bi n d i n g c h a r a c t e r i s t i c s , e i t h e r through conformational changes or by p o l y m e r i s a t i o n and/or denaturation could be caused by e f f e c t s of pressure or s t i r r i n g s t r e s s on the p r o t e i n molecule. U n l i k e p r o t e i n s , nonionic s u r f a c t a n t s are very s t a b l e , and i t i s q u i t e u n l i k e l y that the observed displacement of the bindi n g curves i n the d i a f i l t r a t i o n s t u d i e s i n v o l v i n g benzoic acid-cetomacrogol i n t e r a c t i o n could be caused due to conformational changes i n the s u r f a c t a n t molecules. Palmer (1972) observed changes i n the volume of macromolecule i n the c e l l during d i a f i l t r a t i o n s t u d i e s . S i m i l a r observations were made i n t h i s work. This i s due to an i n i t i a l drop of c e l l pressure because of the l a r g e d i f f e r e n c e i n the volumes of r e s e r v o i r and the c e l l . A pressure drop i n the c e l l w i l l cause an overflow of p r e s e r v a t i v e s o l u t i o n from the r e s e r -v o i r i n t o the c e l l r e s u l t i n g i n d i l u t i o n of the s u r f a c t a n t s o l u t i o n . The uncorrected value of [M] i n the b i n d i n g c a l c u l a t i o n s w i l l produce a downward displacement of the b i n d i n g curves (Palmer, 1972), as observed i n t h i s work 117 (Fig. 19). This effect is analogous to dilution of surfactant-due to osmosis in the equilibrium dialysis study (C,a). It is suggested that dilution of surfactant during d i a f i l t r a t i o n i s the major cause of the dis-crepancy between the d i a f i l t r a t i o n and the equilibrium dialysis binding results. It i s concluded that the anomalous binding curves obtained using the d i a f i l t r a t i o n technique.are due to inherent technical artifacts of the method. Unless these are understood for each drug-macromolecule system, \ d i a f i l t r a t i o n i s not a suitable technique for studying drug-macromolecule interactions. The d i f f i c u l t i e s in the d i a f i l t r a t i o n method overweigh i t s various advantages over the more commonly used techniques, such as equilibrium dialysis. Therefore, the d i a f i l t r a t i o n method was not used further for studying preservative-surfactant interactions. Although equilibrium dialysis i s a slow technique and requires numerous separate experiments to f u l l y characterize the binding curve, i t is thermodynamically sound and, i f carefully used, the binding results are free from most o.f the technical artifacts. D. Interaction of Preservative Mixtures with Cetomacrogol The preservative combinations were selected on the following grounds. 1. Preservative combinations in which both the preservatives have weak association with the surfactant molecule, e.g., benzoic acid and sorbic acid. 2. Preservative combinations in which both the preservatives have strong association with the surfactant molecule, e.g., chlorocresol and propyl paraben. 118 3. Preservative combinations in which one preservative has weak association and the other has strong association with the surfactant molecule, e.g., chlorocresol and methyl paraben; chloroxylenol and methyl paraben. Figure 20 is a Scatchard plot for the interaction of chlorocresol with cetomacrogol in the absence and presence of a constant concentration of methyl paraben. Increasing the concentration of methyl paraben results in a downward displacement of the binding curve (curves B and C). This indicates that there is a possible competition between the chlorocresol and methyl paraben for the same binding sites on a surfactant molecule in a micelle. The experimental data are indicated by points, while the solid lines are theoretical. . Curve A was calculated using n and K values for chlorocresol computed from Equation 25. Curves B and C were generated by substituting the values of n, K^ , K c (obtained from independent binding studies for the individual preservatives) and the experimental [D^] and [C^] into Eq.30 (see appendix 3). The same method was used for calculating the theoretical binding curves shown in Figures 21, 23, 24 and 26. Figure 20 shows reasonable agreement between the theoretical and the experimental values, particularly in view of the fact that small normal variations in the values of the four association constants in Eq.30 can have rather marked influences on the shape and position of a theoretically generated plot. Figure 21 is similar to Fig. 20, and shows the inhibition of the binding of chlorocresol with cetomacrogol in the presence of a constant concentration of propyl paraben. Curve B'.iis a theoretical binding curve for the competitive interaction (Eq.30), whereas curve B is obtained experi-mentally. No correlation was obtained between the experimental and the -119 101 3 I I I —I I J 0.4 0.8 1.2 1.6 2.0 r F i g . 20. Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol i n absence and presence of methyl paraben. Cetomacrogol concentration = 7.69 x 1 0 - 3 moles 1~1. Methyl paraben c o n c e n t r a t i o n : 0, 0 . 0 ;Q , 8.54 x 10" ; Q , 13.14 x 10" 3 moles 1 _ ± . P o i n t s experimental, curves B and C c a l c u l a t e d using Eq.30. 21 L I I • i 0 0.4 0.8 1.2 .1.6 2.Q r F i g . 21. Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol i n absence and presence of p r o p y l paraben. Cetomacrogol concentration = 7.69 x 1 0 - 3 moles l - ^ . P r o p y l paraben concentration: O > > 8.3 x 1 0 - 3 moles l - x . P o i n t s experimental, curve B' c a l c u l a t e d using Eq.30. Binding parameters f o r p r o p y l paraben derived from F i g . 22. 121 122 t h e o r e t i c a l curves. Figure 23 shows the i n t e r a c t i o n of methyl paraben w i t h c e t o -macrogol i n the absence and presence of a f i x e d c o n c e n t r a t i o n of c h l o r o -c r e s o l . Values of n and K f o r methyl paraben alone were obtained from F i g . 22. Increasing the c o n c e n t r a t i o n of c h l o r o c r e s o l r e s u l t s i n downward displacement of the b i n d i n g curve (curves B and C). However, u n l i k e Figure 20, i n c r e a s i n g the c o n c e n t r a t i o n of competitor r e s u l t s i n a decrease i n the value of n and an increase i n the value of K. Since i n a simple competition, of the type described by Eq.30, n should remain constant and K changes, F i g . 23 suggests that the competition between methyl paraben and c h l o r o c r e s o l f o r the b i n d i n g s i t e s i n cetomacrogol molecule i s of a complex nature. Disagreement between the experimental (curves B and C) and the t h e o r e t i c a l (curves B' and C') b i n d i n g curves supports t h i s view. S i m i l a r r e s u l t s were obtained f o r the i n t e r a c t i o n of methyl paraben w i t h cetomacro-g o l i n the presence of c h l o r o x y l e n o l ( F i g . 24, values of n and K f o r c h l o r o -x y l e n o l alone were obtained from F i g . 25) and the i n t e r a c t i o n of benzoic a c i d w i t h cetomacrogol i n the presence of s o r b i c a c i d ( F i g . 26, values of n and K f o r s o r b i c a c i d were obtained from F i g . 27). C o r r e l a t i o n between the t h e o r e t i c a l and the experimental values w i l l be obtained i f the f o l l o w i n g c o n d i t i o n s are observed: I The p r e s e r v a t i v e and the competitor share e x a c t l y the same locus of s o l u b i l i z a t i o n i n the s u r f a c t a n t m i c e l l e . I I I n t e r a c t i o n of a p r e s e r v a t i v e w i t h the s u r f a c t a n t n e i t h e r a l t e r s the nature nor the number of b i n d i n g s i t e s i n the s u r f a c t a n t m i c e l l e . The f i r s t c o n d i t i o n r e q u i r e s an examination of the locus of 123 16 15 14 F i g . 23. 13 12 r 11 o H X I—I M-l ,Q, 10 Scatchard p l o t f o r the i n t e r a c t i o n of methyl paraben w i t h cetomacrogol i n absence and presence of c h l o r o c r e s o l . Cetomacrogol concentration = 7.69 x 1 0 - 3 moles 1~ . C h l o r o c r e s o l concentration:O> 0.0; D , 1.19 x lO" 3;Q , 2.24 x l O - 3 moles I " 1 . P o i n t s experimental, curves B' and C' c a l c u l a t e d using Eq.30. o. b o B' 0.2 0.4 0.6 0.8 1.0 1.2 F i g . 24. Scatchard p l o t f o r the i n t e r a c t i o n of methyl paraben w i t h . cetomacrogol i n absence and presence of c h l o r o x y l e n o l . Ceto-macrogol concentration = 7.69 x 10 -^ moles l - ^ . C h l o r o x y l e n o l concentration: 0, 0.0;#,8.9 x 10~3 moles 1~1. P o i n t s e x p e r i -mental, curve B' c a l c u l a t e d using Eq.30. Binding parameters f o r c h l o r o x y l e n o l derived from F^.g. 25. 125 126 I I I I I I I I 0 0.2 0.4 0.6 0.8 1.0 .1.2 1.4 r . 26. Scatchard p l o t f o r the i n t e r a c t i o n of benzoic a c i d w i t h ceto-macrogol i n absence and presence of s o r b i c a c i d . Cetomacrogol concentration = 7.69 x 1 0 - 3 moles l - x . Sorbic a c i d _ c o n c e n t r a t i o n = 0, 0.0;O , 4.46 x 1 0 - 3 ; • , 10.7 x 10~ 3 moles l" 1. P o i n t s experimental, curves B' and C' c a l c u l a t e d using Eq.30. Binding parameters f o r s o r b i c a c i d derived from F i g . 27. 127 128 s o l u b i l i z a t i o n of the p r e s e r v a t i v e s , used i n t h i s study, i n the cetomacro-g o l m i c e l l e . The locus of s o l u b i l i z a t i o n g e n e r a l l y depends on the p o l a r i t y of the s o l u t e and the HLB of the s u r f a c t a n t (Corby and Elworthy, 1971a). Jacob £t a l . (1971) demonstrated, u s i n g a NMR technique, t h a t phenol was mainly l o c a t e d i n the polyoxyethylene r e g i o n of the cetomacrogol m i c e l l e . Mulley and Metcalf (1956) suggested from UV spectroscopy s t u d i e s that c h l o r o x y l e n o l was l o c a t e d i n the polyoxyethylene r e g i o n of the s u r f a c t a n t m i c e l l e . Higuchi and Lach (1954) a l s o hypothesized that compounds l i k e phenol would form hydrogen bonds w i t h polyoxyethylene groups of n o n i o n i c macromolecule. P a t e l (1967) stud i e d the i n t e r a c t i o n of a number of phenols, i n c l u d i n g c h l o r o c r e s o l , w i t h cetomacrogol and polysorbate 80. An increased b i n d i n g a f f i n i t y of p-chlorophenol over phenol f o r both macromolecules was a t t r i b u t e d to i t s greater c a p a c i t y to undergo hydrogen bond formation. Thus s u b s t i t u t i o n of a c h l o r i n e atom i n the benzene r i n g increased the proton donating power of phenol. Hence, from these s t u d i e s i t i s apparent that the locus of s o l u b i l i z a t i o n of c h l o r o c r e s o l and c h l o r o x y l e n o l i s l a r g e l y i n the oxyethylene chain and i n v o l v e s hydrogen bonding between the a c i d i c hydrogen of the phenol and the e l e c t r o p h i l i c oxygen of the ethylene oxide chain. Corby and Elworthy (197,1b) i d e n t i f i e d the s i t e s of s o l u b i l i z a t i o n , of p-hydroxybenzoic a c i d and i t s e s t e r s i n a cetomacrogol m i c e l l e using UV, NMR, viscometry and s o l u b i l i t y techniques, p-hydroxybenzoic a c i d was wholly s o l u b i l i z e d deep i n the oxyethylene l a y e r of the m i c e l l e . . E t h y l p-hydroxy-benzoate was s o l u b i l i z e d mostly i n the oxyethylene l a y e r adjacent to the core, w h i l e some s o l u b i l i z a t i o n occurred w i t h i n the core. B u t y l p-hydroxy-benzoate was s o l u b i l i z e d mainly at the oxyethylene-hydrocarbon j u n c t i o n , w i t h 129 the phenyl r i n g i n the oxyethylene r e g i o n and the b u t y l chain i n the core. Some s o l u b i l i z a t e was wholly present i n the core. Thus the locus of s o l u b i l i z a t i o n of, p-hydroxybenzoates depends on the p o l a r i t y of the molecule. Since methyl and p r o p y l parabens f a l l between p-hydroxybenzoic a c i d and b u t y l paraben, i t i s l o g i c a l to assume that methyl paraben w i l l be s o l u b i l i z e d mostly i n the oxyethylene l a y e r w h i l e p r o p y l paraben w i l l be l o c a t e d near or at the oxyethylene-hydrocarbon j u n c t i o n . Donbrow and Rhodes (1964, 1966, 1967) showed from t h e i r UV and NMR spectroscopic s t u d i e s that b e n z o i c . a c i d was l o c a t e d at the j u n c t i o n of the hydrocarbon nucleus and the p a l i s a d e l a y e r (ethylene oxide chain) of the cetomacrogol m i c e l l e , w i t h the l i p o p h i l i c benzene r i n g enclosed w i t h i n the nucleus and the h y d r o p h i l i c c a r b o x y l i c a c i d group p r o t r u d i n g i n t o the p a l i s a d e l a y e r . According to these authors, benzoic a c i d so l o c a t e d would l a c k m o b i l i t y because of the presence of the polyethylene oxide chain of the s u r f a c t a n t molecules. The l o c a t i o n could a l s o a l l o w hydrogen bond formation between the a c i d i c hydrogen atom of benzoic a c i d and the e l e c t r o p h i l i c ether oxygen atom of the innermost p a l i s a d e l a y e r of the•cetomacrogol m i c e l l e . Since s o r b i c a c i d l a c k s a phenyl r i n g and i s l e s s l i p o p h i l i c than benzoic a c i d , i t w i l l be p r i n c i p a l l y l o c a t e d i n the p a l i s a d e l a y e r . The i n t e r a c t i o n between s o r b i c a c i d and nonionic macromolecules, such as polysorbate 80 and cetomacro-g o l , i n v o l v e s hydrogen bond formation between a c i d i c hydrogen and the e l e c t r o -p h i l i c oxygen of the polyoxyethylene l a y e r (Blaug and Ahsan, 1961a). Thus the locus of s o l u b i l i z a t i o n of benzoic a c i d and s o r b i c a c i d i n the cetomacro-g o l m i c e l l e i s not e x a c t l y the same. I t i s concluded that none of the p r e s e r v a t i v e combinations used i n t h i s study have e x a c t l y the same locus of s o l u b i l i z a t i o n i n the cetomacrogol 130 m i c e l l e . Thus simple competition, as described by Eq.30, i s not p o s s i b l e . Another p o s s i b l e e x p l a n a t i o n f o r the disagreement between the t h e o r e t i c a l and the experimental b i n d i n g curves comes from s t u d i e s concern-ing the e f f e c t of s o l u t e s on m i c e l l a r molecular weight (Mn). As i n d i c a t e d i n the L i t e r a t u r e Survey (B,e), v a r i o u s authors have demonstrated that the i n t e r a c t i o n of s o l u t e s w i t h s u r f a c t a n t s can cause m i c e l l a r p e r t u r b a t i o n r e s u l t i n g i n a change of m i c e l l a r molecular weight (Mn). A change i n Mn can be due to a change i n the aggregation number of the m i c e l l e s , or i n c l u s i o n of s o l u b i l i z a t e i n t o the m i c e l l e ; Changes i n the aggregation number of a m i c e l l e w i l l ;affect the t o t a l number of bindi n g s i t e s a v a i l a b l e f o r the i n t e r a c t i o n . These.-istudies were made using a s i n g l e s o l u t e . Hence, the complexity i n v o l v e d i n i n t e r p r e t i n g the e f f e c t s of more than one s o l u t e on Mn i s r e a d i l y apparent. In t h i s work, curvature i n the Scatchard p l o t , f o r the i n t e r a c t i o n of a p r e s e r v a t i v e w i t h the nonionic s u r f a c t a n t , was assumed to be due to the exis t e n c e of two e n t i r e l y d i f f e r e n t c l a s s e s of b i n d i n g s i t e s i n the s u r f a c t a n t molecule of a m i c e l l e , that both s i t e s were independent and that s i t e s w i t h i n the c l a s s were e q u i v a l e n t . However, a curvature i n the Scatchard p l o t may a l s o be due t o , what biochemists term, negative c o o p e r a t i v i t y , i . e . , uptake of s o l u t e w i t h i n the m i c e l l e , which p r o g r e s s i v e l y a l t e r s the i n t e r a c t i o n between the b i n d i n g s i t e s and the s o l u t e and leads to a change i n both the number of s i t e s a v a i l a b l e f o r i n t e r a c t i o n and a decrease i n the a s s o c i a t i o n constant. In a d d i t i o n to the f a c t o r s discussed above, the observed d i s p l a c e -ment of b i n d i n g curves ( F i g s . 20, 21, 23, 24 and 26) may be due to cooperative or " a n t i c o o p e r a t i v e " i n t e r a c t i o n s between two p r e s e r v a t i v e s . 131 Although the reasons for the disagreement between the t h e o r e t i c a l and the experimental r e s u l t s are not c l e a r , these findings may have considerable p r a c t i c a l s i g n i f i c a n c e . For example, reduction i n the degree of i n t e r a c t i o n of one or both components of a preservative mixture may r e s u l t i n an increased free concentration of preservative, for a given concentration added, and consequently i n enhanced a n t i m i c r o b i a l a c t i v i t y . The net r e s u l t would be an apparent synergism between the two preservatives. On the other hand, i f the extent.of binding of one or both compounds i s increased, the free concentration would then be reduced. Consequently, the preservative a c t i v i t y would be l e s s than that anticipated from binding studies of the i n d i v i d u a l component. E. Interaction of Chlorocresol with Mixtures of Some Nonionic Surfactants Figure 28 i s a Scatchard p l o t f or the binding of chlorocresol with Texofor A^g (curve A), Texofor A^Q '(curve F) andvvarious mixtures of Texofor A^g and Texofor A^Q (curves B to E ) . Curves A and F are independent of surfactant concentration. The values of the binding parameters (n's and K's) for curve F are of greater magnitude than curve A. An increase i n the value of n indicates that an increase i n the ethylene oxide chain r e s u l t s i n an increase i n the number of binding s i t e s i n the surfactant molecule. This suggests that the locus of s o l u b i l i z a t i o n of c h l o r o c resol i s l a r g e l y assoc-^ ia t e d with ethylene oxide chain of the surfactant molecule. An increase i n K shows that an increase i n ethylene oxide chain length r e s u l t s i n an increase i n the extent of s o l u b i l i z a t i o n of preservative per mole of surfactant. The binding curves f o r Texofor A ^ and Texofor A^Q mixtures are independent of surfactant concentration only when the molar r a t i o of Texofor A., ^  to Texofor 132 CM I o M-l 11 10 JL 0.4 0.8 1.2 r 1.6 2.0 F i g . 28. Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h Texofor A.,, Texofor A--, and mixtures of Texofor A., , and Texofor A,». 16' 60 16 60 A-Texofor A^^: concentration of Texofor A^g (moles l-"*") :0 » 10.57 x 10~ 3; , 31.71 x 1 0 " 3 ; • , 52.85 x 10 . B to E - Texofor A,, lb +Texofor A.-.: concentration of Texofor A.. and A.... i n a mixture ,60 „ 16 _, 60 (moles 1 ) : * , 50.89 x 10 , A l g + 2.75 x 10 , A 6 Q ; # , 10.57 x 10~ 3 A l 6 + 2.75 x 10" 3, A 6 ( );#, 10.57 x 10~ 3, A ± e + 13.26 x 10~ 3, A 6 Q ; 3 , 5.28 x 10~ 3, A l g + 6.63 x 10~ 3, ; • , 2.11 x 10~ 3, A 1 6 + 5.51 x 10~ 3, A,„. F - Texofor Ag f f;"\concentration ro^'-.Texofor' A g Q .-3 Cmoles 1 1 ) :0» 2.75 x 10 3 ; A , 8.26 x 10~ 3; • , 13.77 x 10 133 AgQ was kept constant (curve D). Increasing the molar r a t i o of Texofor A^g to Texofor A^Q r e s u l t s i n a s h i f t of the bi n d i n g curves towards curve A and a consequent decrease i n the values of n and K. S i m i l a r l y , i n c r e a s i n g the molar r a t i o of Texofor A^Q to Texofor A^g r e s u l t s i n the s h i f t of the b i n d i n g curves towards curve F and an increase i n the value of n and K. This i n d i c a t e s that mixing the two s u r f a c t a n t s i n v a r i o u s molar r a t i o s r e s u l t s i n the formation of mixed m i c e l l e s of d i f f e r e n t composition and bi n d i n g c h a r a c t e r i s t i c s . Where i n t e r a c t i o n of a s o l u t e i n v o l v e s a heterogeneous mixture of macromolecules, the value of n i n the Scatchard treatment represents an average of the t o t a l number of b i n d i n g s i t e s of a l l the monomers of v a r y i n g molecular weights (Karush, 1950). Based on t h i s reasoning, the valu e of n f o r the b i n d i n g of c h l o r o c r e s o l w i t h v a r i o u s mixtures of Texofor A-, and Texofor k,n should be the mean of the t o t a l 16 60 number of bi n d i n g s i t e s of Texofor A^g and Texofor A^Q monomers i n a mixed micelle.. F i g u r e 29 i s a Scatchard p l o t f o r the b i n d i n g of c h l o r o c r e s o l w i t h cetomacrogol and polysorbate 80. The bindi n g curves are independent of s u r f a c t a n t c o n c e n t r a t i o n and there i s no s i g n i f i c a n t d i f f e r e n c e between the magnitude of the bi n d i n g parameters f o r cetomacrogol and polysorbate 80. S i m i l a r l y , the b i n d i n g curves of the s u r f a c t a n t mixtures are almost independ-ent of the molar r a t i o of mixing. This i s presumably due to the c l o s e s i m i l a r i t y i n the number of ethylene oxide u n i t s of cetomacrogol and p o l y -sorbate 80s These observations f u r t h e r support the view that the locus of s o l u b i l i z a t i o n of c h l o r o c r e s o l i s l a r g e l y i n the polyoxyethylene r e g i o n of the m i c e l l e . 134 F i g . 29. Scatchard p l o t f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol, polysorbate 80 and mixtures of ceto-macrogol and polysorbate 80. A - Cetomacrogol; concen-t r a t i o n of cetomacrogol (moles 1 : same as given i n F i g . 17. B - Polysorbate 80; co n c e n t r a t i o n of p o l y s o r -bate 80 (moles l " 1 ) : O , 7.64 x 10~ 3;&, 22.93 x 1 0 _ 3 ; -3 Q , 38.22 x 10 . Concentration of cetomacrogol (C) and polysorbate 80 (P) i n a mixture (moles 1 ^ ) : # , 7.69 x 10~ 3, C + 7.64 x 10" 3, P , ; A , 23.11 x 10~ 3, C + 7.64 x 10" 3, P; • , 7.69 x 10~ 3, C +22.97 x 1 0 - 3 , P. 0.4 0.8 1.2 1.6 2.0 135 Figures 30 and 31 show plots of [Dfc] versus [D £] for surfactants mixed in various molar ratios. The curves were calculated by substituting the values of n's and K's, determined experimentally for each surfactant, into Eq.37 and solving for [D^ .] at given [D £] values (see appendix 4). Agree-ment between the calculated curves and the experimental values confirms that 'the binding behavior of surfactant mixtures can be predicted using Eq.37 and the binding parameters of the individual surfactants. f n K [M ] n K [M ] n'K'[M ] n:Kl[M ] 1 ED.] = [D_] I 1 + 1 1 1 + 2 2 1 + 1 1 1 1 + 2 2 1 1 } L 1 + K l L " D f ] 1 + K 2 [ D f ] 1 + K l [ D f ] 1 + K 2 [ D f ] J (Eq.37) The classification of nonionic surfactants in terms of their HLB values is of great practical importance in formulation. Wedderburn (1958) suggested that the binding of a preservative with a nonionic surfactant is a function of the HLB of the surfactant and the physico-chemical properties of the preservative. Equation 37 indicates that the extent of preservative binding in a surfactant mixture is a simple summation of the binding observed using the individual surfactants. Since the HLB values of surfactant mixtures are also obtained by addition of the individual surfactant HLB values, a simple relationship to be expected between HLB values and solute-surfactant binding. HLB values of individual surfactants and surfactant mixtures were calculated using Eqs.57 and 58, respectively (Griffin, 1954). E HLB of a surfactant = y (Eq.57) E E HLB of a surfactant mixture = (f) — -}+ (1-f) -^=- (Eq.58) 136 [D ] x 10 3 moles l " 1 F i g . 30. V a r i a t i o n of f r e e p r e s e r v a t i v e c o n c e n t r a t i o n [D^] w i t h t o t a l preser-v a t i v e concentration [D ] f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h mixtures of Texofor A.,, and Texofor A , n . Concentration of Texofor A, R 16 60 ^ 16 and Texofor A^Q i n a mixture (moles 1 ) : X , 50.89 x 10 , A^g + 2.75 x 10~ 3 A ,-3 60 l60' 10.57 x 10~3, 10.57 x 10 3 A u + 2.75 x 10 A l 6 + 13.26 x 10" , A 6 0 ; B , 2.11 x 10 , A ± 6 + 5.51 x 10 , A 6 Q . P o i n t s experimental, curves c a l c u l a t e d using Eq.37. 137 CD 0) O 6 CM o 2.5 [D ] x 10 3 moles l " 1 F i g . 31. V a r i a t i o n of f r e e p r e s e r v a t i v e concentration [D £] w i t h t o t a l preser-v a t i v e concentration [l> t] f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h mixtures of cetomacrogol and polysorbate 80. Concentration of ceto-7.69 x 10~ 3 C + 7.64 x 10 3 , P; macrogol (C) and Polysorbate 80 (P) i n a mixture (moles 1 ^ ) : , - , 23.11 x 10~ 3, C + 7.64 x 10~ 3, P; -3 -3 7.69 x 10 , C + 22.97 x 10 , P. P o i n t s experimental, curves c a l c u l a t e d using Eq.37. 138 Where: E = Weight per cent ethylene oxide i n a surfactant molecule. E^ = Weight per cent ethylene oxide i n a surfactant molecule of type I. = Weight per cent ethylene oxide i n a surfactant molecule of type I I . f = Fract i o n of a surfactant i n surfactant mixture. Figures 32 and 33 show that there i s a l i n e a r r e l a t i o n s h i p between HLB and [ D t ] , where [ D t ] values were calculated using Eq.37 for given values of [ D r ] , For the i n t e r a c t i o n of chlorocresol with Texofor A.., t l b and AgQ, F i g . 32 shows that an increase i n HLB r e s u l t s i n a decrease i n the value of [D ], i . e . , i n p r a c t i c a l terms, a smaller t o t a l preservative concentration i s required to maintain a given concentration of free preser-v a t i v e i n the aqueous phase as the composition of the surfactant mixture i s changed to ; increase the HLB. Such r e s u l t s i n d i c a t e that when surfactant concentrations are expressed as equivalents of ethylene oxide per cent, there i s an apparent decrease i n the e f f i c i e n c y of s o l u b i l i z a t i o n with increase i n ethylene oxide chain length. Similar binding c h a r a c t e r i s t i c s have been reported f o r the i n t e r a c t i o n of aldehydes with polyoxyethylene ethers of varying ethylene oxide chain length (M i t c h e l l and Wan, 1964), and the i n t e r a c t i o n of benzoic acid d e r i v a t i v e s (Goodhart and Martin, 1962) and barbiturates (Gouda, Ismail and Motawi, 1970) with polyoxyethylene stearates • of varying ethylene oxide chain length.using s o l u b i l i t y techniques. The decrease i n the e f f i c i e n c y of s o l u b i l i z a t i o n with increase i n the number of ethylene oxide units was explained (M i t c h e l l and Wan, 1964) i n terms of a decrease i n the m i c e l l a r molecular weight and i n the number of surfactant molecules per m i c e l l e as the ethylene oxide chain i s lengthened. In contrast to. Texofors A.,and A A n , the i n t e r a c t i o n of chlorocresol 139 16 14 12 10 CO CU O a ro O 14 15 16 17 18 19 HLB F i g . 32. [D f c] versus HLB at constant [D f] f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h Texofor A. 16 1 Texofor A^Q and mixtures, of Texofor A^g and Texofor AgQ. T o t a l c oncentration of s u r f a c t a n t or s u r f a c t a n t mixtures = 1% (w/v). Concentration of f r e e c h l o r o c r e s o l [D ] : A, 1.0 x 10~ 3; -3 -3 -1 B, 2.0 x 10 ; C, 3.0 x 10 moles 1 . HLB Texofor = 14.88; HLB Texofor A,„ = 18.67. 60 Curves c a l c u l a t e d using Eq.37, 140 CO <D rH O S3 co o 13 Fig. 33. 14 15 16 HLB [D^Jversus HLB at constant [D^] for the interaction of chlorocresol with cetomacrogol, polysorbate 80 and mixtures of cetomacrogol and polysorbate 80. Total concentration of surfactant or surfactant mixture = 1% (w/v). Concentration of free chlorocresol [D^]: A, 0.5 x 10~3; B, 1.0 x 10~3; C, 1.5 x 10~3 moles l " 1 . HLB ceto-macrogol = 15.57. HLB polysorbate 80 = 13.46. Curves calculated using Eq.37. 141 w i t h cetomacrogol, polysorbate 80 and mixtures of cetomacrogol and p o l y -sorbate 80 i s v i r t u a l l y independent of HLB f o r a given [ D £ ] , F i g . 33. Since cetomacrogol and polysorbate 80 have approximately the same number of ethylene oxide u n i t s , F i g . 33 supports the suggestion that s o l u b i l i z a -t i o n i s a ssociated e s s e n t i a l l y w i t h the ethylene oxide chain. Kazmi and M i t c h e l l (1971) described a three-chambered d i a l y s i s method f o r determining the d i s t r i b u t i o n of a p r e s e r v a t i v e i n an emulsion and discussed the advantages of t h i s d i r e c t approach over determining the necessary f a c t o r s f o r s u b s t i t u t i o n i n t o a mathematical model. One of the problems w i t h the model approach i s that most emulsions c o n t a i n more than one s u r f a c t a n t and the degree of b i n d i n g w i l l depend on the nature and composition of the s u r f a c t a n t mixture. However, the present work shows that i t i s unnecessary to determine the b i n d i n g parameters f o r each s u r f a c t a n t mixture s i n c e the degree of i n t e r a c t i o n of the mixture w i t h a p r e s e r v a t i v e can be p r e d i c t e d from a knowledge of the parameters." of the i n d i v i d u a l s u r f a c t a n t s comprising the mixture. F. D i s t r i b u t i o n of C h l o r o c r e s o l i n L i q u i d Paraffin-Water Systems Figure 34 shows the concentration of c h l o r o c r e s o l i n the o i l phase [ D q ] , p l o t t e d as a f u n c t i o n of the concentration i n the aqueous phase [ D £ ] . A l i n e a r r e l a t i o n s h i p between and [D £] i n d i c a t e s that the d i s t r i b u t i o n of c h l o r o c r e s o l between l i q u i d p a r a f f i n and water obeys the simple p a r t i t i o n law. The slope of the l i n e gives the p a r t i t i o n c o e f f i c i e n t , K°. The value of K° obtained from the slope was 1.67 which i s c l o s e to the value of 1.53 w reported i n the l i t e r a t u r e (Bean et a l *., 1965). A small discrepancy between the observed and the l i t e r a t u r e values of K° may be due to the f a c t w J that l i g h t l i q u i d p a r a f f i n v a r i e s from one batch to another. 142 2.0 1.6 1.2 0.4 [D f] g . l . " 1 F i g . 34. Concentration of p r e s e r v a t i v e i n o i l , [D^l> a s a f u n c t i o n of f r e e p r e s e r v a t i v e i n the aqueous phase, [D^] f o r the d i s t r i b u t i o n of c h l o r o c r e s o l between l i q u i d p a r a f f i n and water. P o i n t s experimental, l i n e f i t t e d using l e a s t squares method. Slope = 1.67. 143 G. Calculation of Total Concentration of Chlorocresol Required in Cetomacrogol Solutions to Produce the Desired Concentration of Free Preservative in the Aqueous Phase Figure 35 shows the interaction of chlorocresol with various concentrations of cetomacrogol (derived from Fig. 17, silicone rubber data) represented in the form of ED t] versus [D^]. There i s close agreement between the experimentally determined values and the curves predicted using Eq.28. The values of the binding parameters (n's and K's) for the sub-stitution into Eq.28 were derived in a similar manner to that described earlier (C(a)2). f n K [M] n K M 1 [ D j = [D.] I 1 + 1 1 - + -2-2 } L 1 + ^ [ D f ] 1 + K 2[D f] J (Eq.28) Hence, Eq.28 is. valid and enables a calculation to be made of the total concentration of preservative required in a surfactant solution to produce the desired concentration of free preservative in the aqueous phase. H. Calculation of the Concentration of Chlorocresol Required in a Liquid Paraffin Emulsion to Produce the Desired Concentration of Free Preservative in the Aqueous Phase (a) Estimation of free chlorocresol concentration in a liquid paraffin emulsion Analysis of the concentration of chlorocresol in the aqueous compartment of the two-chambered dialysis c e l l enables the free chlorocresol concentration, [D^], to be determined. From [D^], amount of chlorocresol bound to the dialysis membrane (Fig. 36) and the total amount of chlorocresol F i g . 35. V a r i a t i o n of f r e e p r e s e r v a t i v e concentration D>f], w i t h t o t a l p r e s e r v a t i v e concentration, [D ] , f o r the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol. Cetomacrogol concentrations (%):0» 1-0; ^7 > 2.0; • , 3.0; O , 5.0; A , 10.0. P o i n t s e x p e r i -mental, curves c a l c u l a t e d using Eq.28. [D f] x 10 g . l . -1 145 F i g . 36. Binding of c h l o r o c r e s o l w i t h s i l i c o n e membrane. '. P o i n t s experimental, l i n e f i t t e d using l e a s t squares method. 146 added, £ C^ g] + [D^] Jcan be determined, although i t i s not p o s s i b l e to separate these q u a n t i t i e s . Where i t i s d e s i r a b l e to d i f f e r e n t i a t e between [D^] and [ 0 ^ ] , a three chambered d i a l y s i s technique, as described by Kazmi and M i t c h e l l (1971b), can be used (see page 62 and 141), (b) F i g u r e 37 i s a p l o t of the t o t a l p r e s e r v a t i v e c o n c e n t r a t i o n , [ D ] , versus the co n c e n t r a t i o n of f r e e p r e s e r v a t i v e i n the aqueous phase, [D^], f o r She d i s t r i b u t i o n of c h l o r o c r e s o l between l i g h t l i q u i d p a r a f f i n - w a t e r - c e t o -macrogol systems of a f i x e d o i l - w a t e r r a t i o . There i s c l o s e agreement between the experimentally determined values and the curve p r e d i c t e d using Eq.48. Close agreement was a l s o found between [D^] p r e d i c t e d using Eq.48 and the observed values f o r the d i s t r i b u t i o n of c h l o r o c r e s o l between l i q u i d paraffin-water-cetomacrogol systems of v a r y i n g o i l - w a t e r r a t i o s (Table 6 ). Thus, equation 48 i s v a l i d and permits a c a l c u l a t i o n to be made of the t o t a l c o n c e n t r a t i o n of the p r e s e r v a t i v e r e q u i r e d i n an emulsion to provide the d e s i r e d c o n c e n t r a t i o n of f r e e p r e s e r v a t i v e i n the aqueous phase. [D] = j c D ^ l + v n ^ M / a + K j [ D f ] ) + n 2 K 2 [ M ] / ( l + K ^ ] ) + K°qJ|/(q + 1) (Eq.48) I . C o r r e l a t i o n of Physico-Chemical Data w i t h A n t i m i c r o b i a l A c t i v i t y In the previous s e c t i o n s (G and H), physico-chemical models f o r p r e d i c t i n g the re q u i r e d p r e s e r v a t i v e concentration i n s u r f a c t a n t s o l u t i o n s • and emulsions were eva'Iuj.ted*. The i m p l i c i t assumption underlying the development of these models i s that a n t i m i c r o b i a l a c t i v i t y i s l a r g e l y a f u n c t i o n of the conc e n t r a t i o n of f r e e p r e s e r v a t i v e i n the aqueous phase and that p r e s e r v a t i v e bound to s u r f a c t a n t m i c e l l e s or p a r t i t i o n e d i n t o the o i l 147 L 1 1 1 I I 1 2 3 . 4 5 [D F] x 10 3 moles l " 1 F i g . 37. V a r i a t i o n of f r e e c h l o r o c r e s o l c o n c e n t r a t i o n , [D £ ] , i n the aqueous phase of the emulsion w i t h t o t a l c h l o r o c r e s o l , [D], f o r anO/W emulsion c o n t a i n i n g 50% v/v l i q u i d p a r a f f i n e m u l s i f i e d w i t h 3% w/v cetomacrogol. P o i n t s experimental, curve c a l c u l a t e d using. Eq.48. 148 TABLE 6. Chlorocresol Concentration i n L i q u i d P a r a f f i n Emulsions S t a b i l i z e d with Cetomacrogol Oil/Water r a t i o Moles I , " 1 x 10 3 Standard D e v i a t i o n 105 I D ] C a l c u l a t e d u s i n g Eq.48 V>t3 Required P f ] Observed* 0.2 27.15 2.45 2.45 8.27 0.5 22.54 2.45 2.32 3.53 1.0 17.93 2.45 .2.35 18.2 *Mean of three observations. -3 Cetomacrogol con c e n t r a t i o n = 23.1 x 10 moles JL, 149 phase i s b i o l o g i c a l l y i n a c t i v e . Good co r r e l a t i o n s were obtained between the predicted and the observed values of free preservative concentration i n the aqueous phase, [D^], using Eq.28 for surfactant solutions and Eq.48 for emulsions. The present work i s an attempt to c o r r e l a t e the physico-chemical data with the observed an t i m i c r o b i a l a c t i v i t y i n order to evaluate the v a l i d i t y of the hypothesis that a n t i m i c r o b i a l a c t i v i t y i s l a r g e l y a function of the concentration of free preservative and i s e s s e n t i a l l y independent of factors such as surfactant concentration and oil-water r a t i o . The death rate of E_. c o l i was taken as a measure of preservative a c t i v i t y . . (a) Comparison between M i l l i p o r e and Pour-Plate Techniques The death rate of micro-rorganisms i n a given s o l u t i o n i s generally studied by counting the number of surviving organisms as a function of time. Pour-plate technique or r o l l tube method are generally used f o r the v i a b l e counting of micro-organisms. These techniques involve d i l u t i o n of the sample i n normal s a l i n e or H strength Ringer's s o l u t i o n and subsequent p l a t i n g on agar. A few d i f f i c u l t i e s a r i s e with the use of these techniques fo r studying the death rate of micro-organisms i n systems containing preser-v a t i v e s . Large d i l u t i o n s are required to reduce the concentration of the preservative which would otherwise i n h i b i t the growth of micro-organisms upon p l a t i n g . This i s a serious l i m i t a t i o n , e s p e c i a l l y when the death rate i s followed up to 100% m o r t a l i t y . Since at high m o r t a l i t y l e v e l s large d i l u t i o n s are not possible, the chances of carry over of preservative to the growth medium are great. The M i l l i p o r e method obviates these problems by r i n s i n g the test f i l t e r with s t e r i l e f l u i d a f t e r sample f i l t r a t i o n . In 150 the Millipore-procedure, the test f i l t e r i s a s p e c i a l type having a hydrophobic rim approximately 3 millimeters wide. This prevents i n t r u s i o n of the sample f l u i d into the area under the sealing edge of the f i l t e r holder. No matter how large a sample i s used, a l l growth-inhibiting r e s i d u a l s are within immediate reach of the r i n s e f l u i d , and are d i l u t e d to the point where they have no discoverable e f f e c t on c u l t u r i n g . Thus, using the M i l l i p o r e technique, i t i s possible to follow the death rate to any desired l e v e l of mortality without l i m i t i n g the sample s i z e of without any danger of r e s i d u a l preservative i n h i b i t i n g the growth of micro-organisms. Figure 38 shows that there was a good c o r r e l a t i o n between the M i l l i p o r e method and the pour-plate technique for 'the enumeration of IS. c o l i . Thus the M i l l i p o r e technique gave v i a b l e counts comparable with the w e l l established pour-plate method. (b) B a c t e r i c i d a l A c t i v i t y of Chlorocresol i n Water Figure 40 i s a p l o t of the logarithm of the number of organisms surviving versus time (semi log p l o t ) for the b a c t e r i c i d a l a c t i v i t y of chlorocresol i n water against IS. c o l i . For a given chlorocresol concentration a c u r v i l i n e a r r e l a t i o n s h i p , instead of s t r a i g h t l i n e , was obtained, This suggests that the death rate of IS. c o l i i n the aqueous ch l o r o c r e s o l solutions does not follow f i r s t order k i n e t i c s . Similar r e l a t i o n s h i p s were obtained for the death rate of IS. c o l i i n solutions of c h l o r o c r e s o l i n ceto-macrogol and l i q u i d p a r a f f i n emulsions. However, when the same data i s represented i n the form of p r o b i t % survivors versus time p l o t (probit p l o t ) , a s t r a i g h t l i n e r e l a t i o n s h i p (Fig./ 41) r e s u l t s . This means that the e x t i n c t i o n rate of E . c c o l i follows a normal d i s t r i b u t i o n . PLATE COUNT x 10 -2 i g . 38. Comparison between M i l l i p o r e f i l t r a t i o n and pour-plate techniques f o r the enumeration of E_. c o l i . D i f f e r e n t symbols represent separate experiments. C o r r e l a t i o n c o e f f i c i e n t = 0.95. 152 B I J i I i i i ' ' 0 1 . 2 3 4 5 6 7 8 HOURS. Fig. 39. Semilogarithmic plot of the number of organisms/ml as a function of time for the survival of E_. coll: in,. A - water, B - aqueous cetomacrogol solutions: concen— tration of cetomacrogol as in Fig. 40, C — liquid paraffin emulsions: values- of q and [M ] as in Fig . 45. 153 CD 6 CD •r l c n) 60 u O <+-l o u CD •§ 10" 10" 10" Fig. 40. Semilogarithmic plot of the number of organisms/ml as a function of time for the bactericidal activity of chlorocresol i n water against E_. c o l i . Chlorocresol con-centration (%): • , 0.02;O , -f, 0.035. 10' _L _L J 8 3 4 5 HOURS 154 HOURS .g. 41 . P r o B i t % s u r v i v o r s as a funct ion , of t ime f o r the b a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n water aga ins t E. c o l l . C h l o r o c r e s o l concen t r a t i on ( % ) : • , 0 .02 ; Q , 0 .03 ; X , 0 .035 . ' 155 Probit p l o t s o f f e r many advantages over semilog p l o t s , e.g., a s t r a i g h t l i n e i s easier to f i t to a set of points than a nonlinear r e l a t i o n s h i p ; i t i s easier to perform s t a t i s t i c a l analyses of the data; the slope ofpprobit p l o t seems to be l i t t l e influenced by small changes i n the concentration of organisms i n the inoculum (Kavanagh, 1963), whereas a small change i n the inoculum s i z e produces marked change i n the slope of a semilog p l o t ; extrapolation of the s t r a i g h t l i n e i n the probit p l o t can give information about the time required to achieve 100% m o r t a l i t y with much more accuracy than extrapolation of the curve i n the semilog p l o t . Thus, i n the present work the probit p l o t i s considered as a method of c h o i c e f f o r the graphical presentation of the data on the death k i n e t i c s of E_. c o l i i n aqueous solutions, surfactant solutions and l i q u i d p a r a f f i n emulsions containing c h l o r o c r e s o l . Figure 41 i s a p l o t of the probit % survivors versus time for the b a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n water against _E. c o l i . The symbols represent experimental data, while l i n e s were f i t t e d using Wang c a l c u l a t o r programmed for l i n e a r regression analysis (GLICKSTAT, designed by Dr. G l i c k , Department of Mathematics, U.B.C). Increasing the concentration of c h l o r o c r e s o l from 0.02% to 0.03% makes l i t t l e change i n the death rate of E_. c o l i . However, at a concentration of 0.035% c h l o r o c r e s o l , there i s a marked increase i n the death rate. Since 0.035% c h l o r o c r e s o l produced 99% m o r t a l i t y within 6-8 hours, a time s u i t a b l e from the viewpoint of experi-mental design, t h i s concentration was selected as a reference standard to which the b a c t e r i c i d a l a c t i v i t y of c h l o r o c resol i n aqueous cetomacrogol solutions and l i q u i d p a r a f f i n emulsions could be compared (see sections c and d). The r e s u l t s of the c o n t r o l experiments are shown i n F i g . 39(A). 156 The v i a b i l i t y of E_. c o l i i n water remained almost unchanged during the experimental period of eight hours. (c) B a c t e r i c i d a l A c t i v i t y of Chlorocresol i n Aqueous Solutions of Cetomacrogol Figure 42 shows probit % survivors plotted as a function of time for the b a c t e r i c i d a l r . a c t i v i t y of chlorocresol i n water (curve A) and aqueous solutions of cetomacrogol against IS. c o l i . I'The aqueous solution contained 0.035% chlorocresol and the surfactant solutions contained s u f f i c i e n t t o t a l preservative, calculated from Eq.28, to provide a free concentration of 0.035% chlorocresol i n the aqueous phase. The symbols represent experimental data andithe l i n e s were f i t t e d using the l i n e a r regression analysis program. The slopes of the l i n e s i n Fig. 42 were compared using t tests, without assumption of equal variances (Eq.59), and using modified Welch degrees of freedom (Eq.60). Test of significance between the two slopes were performed using the n u l l hypothesis of equality against a one sided alternative. r b l " b 2 t = _ _ _ _ _ (Eq.59) where b^ and = slopes of curves 1 and 2 respectively. 2 2 S, and S, = variance of slopes of curves 1 and 2 respectively. D l . D2 — £~--g£ (iq.60) D.F. Nj_ - 2 N 2 - 2 157 HOURS ig. 42. Probit % survivors as a function of time for the bactericidal activity of chlorocresol in aqueous cetomacrogol solutions against E. c o l i . Cetomacrogol concentration (%): A, 0.0 (from Fig. 41); B . O , 1.0; C , D , 3.0; D, A, 5.0. Total preservative concentration [D ]%: A, 0.035; B, 0.1743; C, 0.4528; D, 0.7314. I n i t i a l free preservative concentration, [D f], for a l l solutions = 0.035 %. Points experimental, lines fitted using least squares method. 158 where D.F. = modified Welch degrees of freedom. and N 2 = Number of observations f o r curves 1 and 2 r e s p e c t i v e l y . 9 = i ( S b V V + ( S b 2 / N 2 > Table 7 gives the summary of the s t a t i s t i c a l a n a l y s i s of the slopes of the curves i n F i g . 42. Curves B, C and D ( s u r f a c t a n t s o l u t i o n s ) are s i g n i f i c a n t l y d i f f e r e n t from curve A (water). Curves C and D (3% and 5% cetomacrogol s o l u t i o n s r e s p e c t i v e l y ) are s i g n i f i c a n t l y d i f f e r e n t from Curve B (1% cetomacrogol s o l u t i o n ) . However, the d i f f e r e n c e between curves C and D i s n o n s i g n i f i c a n t . Since the aqueous and cetomacrogol s o l u t i o n s have the same i n i t i a l [D^] and only [D f c] v a r i e s , a s i g n i f i c a n t d i f f e r e n c e between the slopes of the s u r f a c t a n t and aqueous curves suggests that the a n t i m i c r o b i a l a c t i v i t y i n cetomacrogol s o l u t i o n s i s perhaps not simply a f u n c t i o n of the concen-t r a t i o n of p r e s e r v a t i v e i n the aqueous phase, and that some a d d i t i o n a l f a c t o r s may be in v o l v e d i n c o n t r o l l i n g the a n t i b a c t e r i a l a c t i v i t y . The r e s u l t s and s t a t i s t i c a l a n a l y s i s appear to show t h a t : (1) the p r o b i t - s u r v i v o r curves f o r the s u r f a c t a n t s o l u t i o n s are s i g n i f i c a n t l y d i f f e r e n t from those i n water. (2) the slope of the p r o b i t - s u r v i v o ^ , curve i s not independent of s u r f a c t a n t c o n c e n t r a t i o n . In view of these f i n d i n g s i t i s necessary to examine the assumption u n d e r l y i n g both the development of the mathematical models and TABLE 7. Comparison of Slopes in Fig. 42 Using t Test. Curve t Calculated using Eq.59 Modified Welch Degrees of Freedom (Eq.60) i' i Probability Comparison of curves B C, and D with curve A B 2.47 27.62 0.01 C 6.12 29.86 < 0.005 D 5.23 30.42"" <C 0.005 Comparison of curves C and D with curve B • C 3.77 43.75 < 0.005 D 2.93 43.29 < 0.005 Comparison of curve C with curve D r. C 0.70 43.88 < 0.3 Ln VO 160 the exper imenta l d e s i g n . (1) Comparison between Aqueous and Su r fac tan t So l u t i ons of P r e s e r v a t i v e ' A l though there i s a s i g n i f i c a n t d i f f e r e n c e between the s lopes of the aqueous and s u r f a c t a n t cu rves , comparison w i t h F i g . 41 shows that a l l the s u r f a c t a n t curves f a l l between the va lues of the s lopes f o r 0.030% and 0.035% aqueous c h l o r o c r e s o l s o l u t i o n s , i . e . , a r e l a t i v e l y sma l l d i f f e r e n c e i n [D £ ] w i l l produce a s i g n i f i c a n t change i n the s l o p e . Th is concen t r a t i on d i f f e r e n c e should be compared w i t h the l a r g e d i f f e r e n c e between L^^l f ° r each s u r f a c t a n t s o l u t i o n , i . e . , B, 0.1743%; C, 0.4528%; D, 0.7324%. I t i s apparent from F i g . 41 that the s lope of the p r o b i t - s u r v i v o r curve i s ve ry s e n s i t i v e to changes i n [ D £ ] , i n the concen t r a t i on range chosen f o r these exper iments . The re fo re , any f a c t o r produc ing a change i n [D £ ] w i l l have a s i g n i f i c a n t e f f e c t on the s lope of the p r o b i t - s u r v i v o r cu rve . Two f a c t o r s may change [D £ ] i n su r f a c t a n t s o l u t i o n s r e l a t i v e to a s o l u t i o n i n wa te r : I. D e p l e t i o n of p r e s e r v a t i v e due to i n t e r a c t i o n w i t h m i c r o -organisms or f o r e i g n , m a t e r i a l s (see Tables I I I and IV i n Bean, 1972)-, v o l a t i l i z a t i o n , chemica l decomposi t ion (Nair and Lach , 1959) , or even metabol ism by the m ic ro-organ i sms (Soko l sk i e t a l . , (1962) w i l l be s i g n i f i -c a n t l y g rea te r from a s o l u t i o n i n water than from a s o l u b i l i z e d system where the m i c e l l e s w i l l ac t as a r e s e r v o i r of p r e s e r v a t i v e . F igu re 43 shows a d i r e c t r e l a t i o n s h i p between, the % l o s s of c h l o r o c r e s o l from the water and the aqueous phase of cetomacrogol (see appendix 5 f o r the c a l c u l a t i o n of t h i s parameter ) , when equa l amounts of c h l o r o c r e s o l are removed from water and the s u r f a c t a n t s o l u t i o n . On the percentage b a s i s , f o r a g iven l o s s i n 161 m o Q) CO si CO 6-2 o di rH 3 O cr ao <! o u a CJ o ed u G o u rH CU O O CO cu u u o ' u o rH .fl o MH o CO CO o r J 50 40 U 30 h 20 10 10 20 30 40 Loss of C h l o r o c r e s o l From -Water (%) 50 F i g . 43. Comparison between percent l o s s of c h l o r o c r e s o l from water and aqueous phase of cetomacrogol when equal amount of p r e s e r v a t i v e i s removed from water and cetomacrogol s o l u t i o n . Cetomacrogol concen-t r a t i o n (%): A, 0.1; B, 0.5; C, 1.0; D, 2.0; E, 3.0; F, 4.0; G, 5.0. 162 the concentration of preservative from water, the lo s s i n the concentration of preservative from the aqueous phase of the surfactant i s smaller and i s a function of the surfactant concentration. This i s apparent from the decrease i n the slope.of curves with increase i n the surfactant concentration. A slope approaching unity means that t h e a a b i l i t y of the system to r e s i s t changes i n [D^] i s decreasing i n the d i r e c t i o n of water which o f f e r s no resistance to change. If the a b i l i t y of a system to r e s i s t changes i n [D^] i s defined as i t s Capacity then the inverse of the slope of a given curve i n F i g . 43 becomes a numerical expression for capacity. Allawala and Riegelman (1953) consider saturation s o l u b i l i t y of a solute i n a given system as a measure of capacity. Therefore, an increase i n s o l u b i l i t y r e s u l t s i n an increase of capacity. B a s i c a l l y , both d e f i n i t i o n s are s i m i l a r because saturation s o l u b i l i t y of the preservative increases with increase i n surfactant concentration. S i m i l a r l y , the inverse of the slope i n F i g . 43 increases with increase i n surfactant concentration (Fig. 44). However, the d e f i n i t i o n of Allawala and Riegelman (1953) i s q u a l i t a t i v e i n nature and does not give a numerical value to capacity. Figure 44 shows a l i n e a r r e l a t i o n s h i p between the capacity and the surfactant concentration, [M], for the i n t e r a c t i o n of c h l o r o c r e s o l with Texofor A ^ , Texofor A|g. (cetomacro-gol) and Texofor Ag^. For a given surfactant concentration the capacity decreases with an increase in,the ethylene oxide chain. This i s because the e f f i c i e n c y of s o l u b i l i z a t i o n decreases with an increase i n the ethylene oxide units (see page U138 ). At zero surfactant concentration the capacity of the system i s equal to 1.0. Thus, from F i g s . 43 and 44, i t i s evident that the capacity of the system i s an important f a c t o r which should be taken i n t o consideration i n any study i n v o l v i n g a comparison of the preservative a c t i v i t y between two unlike systems such as water and surfactant solutions. 163. 164 I I . A second but l e s s l i k e l y f a c t o r which may change i s the adsorption of surfactant monomer on the microbial surface. This w i l l reduce the free surfactant concentration. Re-establishment of the m i c e l l e -monomer equilibrium w i l l lead to the l i b e r a t i o n of some m i c e l l a r preser-v a t i v e , an increase i n [D £] and hence ant i m i c r o b i a l a c t i v i t y . Hence, a comparison of a n t i m i c r o b i a l a c t i v i t y i n surfactant solutions with solutions i n water may lead to confusion unless i t can be shown that [D £] remains the same throughout the experiment. Previous studies have shown, as expected from the capacity concept, that surfactant solutions are s l i g h t l y more a c t i v e than aqueous solutions containing the same [D^] ( M i t c h e l l , 1964; Humphreys, Richardson and Rhodes, 1968; Brown, 1968; Bradshaw, Rhodes and Richardson, 1972). However, i n the present work, the r e s u l t s i n F i g . 42 show that the surfactant solutions are l e s s a c t i v e than the aqueous s o l u t i o n . In view of the d i f f i c u l t i e s which a r i s e i n attempting to compare two unlike systems, such as aqueous and surfactant solutions, i t i s better to compare the a n t i m i c r o b i a l a c t i v i t y of the preservative so'lubilized i n various surfactant concentrations having the same i n i t i a l value of [ D £ ] , Changes i n [D £] due to i n t e r a c t i o n of preservative or surfactant with the micro-organism, or other f a c t o r s , w i l l be r e l a t i v e l y l e s s and any enhancement i or diminution of a c t i v i t y can then be a t t r i b u t e d to the presence of s u r f a c t -ant. This approach was used i n designing the experiments for the present study (Fig. 42). 165 (2) Comparison between Surfactant S o l u t i o n s of P r e s e r v a t i v e A s i g n i f i c a n t d i f f e r e n c e between the slopes of curve B and the slopes of curves C and D suggests that i n c r e a s i n g the conc e n t r a t i o n of cetomacrogol r e s u l t s i n a decrease i n a n t i m i c r o b i a l a c t i v i t y . However, there i s no s i g n i f i c a n t d i f f e r e n c e between the slopes of C and D and t h i s i n d i c a t e s that i n c r e a s i n g the conc e n t r a t i o n of cetomacrogol between 3%-5% produces l i t t l e f u r t h e r change i n a c t i v i t y . The small decrease i n a c t i v i t y w i t h increase i n the cetomacrogol c o n c e n t r a t i o n could be due to the f o l l o w i n g f a c t o r s : I . S t i m u l a t i o n of the growth of E. c o l i by cetomacrogol: S t i m u l a t i o n of the growth of micro-organisms by nonionic s u r f a c t a n t s i s not an uncommon phenomenon. Dubos and Davis (1946) reported that polysorbate 80 encourages the growth of t u b e r c l e b a c i l l i i n c u l t u r e media which they a t t r i b u t e to the surface a c t i v i t y of the nonionic s u r f a c t a n t . Englar (1950) and Wil l i a m s et a l . (1947) reported that nohionics s t i m u l a t e the growth of c e r t a i n micro-organisms, w h i l e B o l l e and Mirimanoff (1950) found that a l l the nonionics they tes t e d (carbowax 1500, some c r i l l s , spans, and tweens)_ possessed a s t i m u l a t i n g a c t i o n on the development of mould mycelia. 'de Navarre and B a i l e y (1956) and de Navarre (1957b) showed that fungal m y c e l i a l growth i n media c o n t a i n i n g nonioni'cs .wasmore l u x u r i a n t than i n p l a i n media. However, i n the present work, the p o s s i b i l i t y of the s t i m u l a t i n g e f f e c t of cetomacrogol on the growth r a t e of E_. c o l i i s r u l e d out because the r e s u l t s of c o n t r o l experiments ( F i g . 39,B) showed a s l i g h t decrease, i n s t e a d of enhancement, of the growth of the organism i n the s u r f a c t a n t s o l u t i o n s . I I . P r o t e c t i o n of E. c o l i by cetomacrogol from the l e t h a l e f f e c t of 166 c h l o r o c r e s o l : The p o s s i b i l i t y of t h i s e f f e c t i s very l i k e l y because there are evidences which suggest that very low concentrations of nonionic s u r f a c t a n t s p r o t e c t micro-organisms from the l e t h a l e f f e c t of a n t i m i c r o b i a l agents. Beckett et a l . (1958a,b; 1959) have shown th a t a d d i t i o n of ceto-macrogol, i n as low asconcentration as p o s s i b l e without a f f e c t i n g complex formation, reduced the uptake of h e x y l r e s o r c i n o l from s o l u t i o n s by jS. c o l i at a l l c o n c e n t r a t i o n l e v e l s of the phenol s t u d i e d . According to these authors, cetomacrogol appeared to prevent or i n t e r f e r e w i t h the i n t e r a c t i o n of phenol w i t h the cytoplasmic membrane of the b a c t e r i a . J u d i s (1962) has a l s o shown th a t the s i t e of a c t i o n of phenolic d i s i n f e c t a n t s i n _E. c o l i i s at the cytoplasmic membrane, and Tween 80 protected IS. c o l i from t h e ' l e t h a l e f f e c t s of c h l o r o x y l e n o l . Wedderburn (1958) observed changes i n Gram-staining c h a r a c t e r i s t i c s of some b a c t e r i a a f t e r contact w i t h n o n i o n i c s . Gram-positive organisms turned Gram-negative a f t e r a p e r i o d of contact w i t h the nonionic and, on sub c u l t u r e , r e v e r t e d back to Gram-positive. S i m i l a r l y Gram-negative organisms upon contact w i t h nonionicqbecame Gram-positive f o r a pe r i o d and, when t r a n s -f e r r e d to media without n o n i o n i c , once again turned Gram-negative. This phenomenon was a l s o observed by N. and A. Delmotte (1956).. In the same study Wedderburn (1958) demonstrated that some organisms, i n c l u d i n g IS. c o l i , were rendered more r e s i s t a n t to c e r t a i n germicides a f t e r contact w i t h n o n i o n i c s . This r e s i s t a n c e v a r i e d not only f o r d i f f e r e n t nonionics but a l s o f o r d i f f e r e n t organisms. These phenomena were s a i d to be due to the p r o t e c t i v e e f f e c t of nonionics on the c e l l w a l l of the micro-organisms. From the observations quoted above i t appears that the p r o t e c t i o n 167 o f f e r e d by nonionics to micro-organisms against the l e t h a l e f f e c t of the a n t i m i c r o b i a l agents i s r e l a t e d to the mechanism of a c t i o n of the p r e s e r -v a t i v e s . Only the e f f e c t of those a n t i m i c r o b i a l agents which exert t h e i r l e t h a l e f f e c t through d i r e c t a c t i o n on the cytoplasmic membrane of the organisms w i l l be i n h i b i t e d . In c o n t r a s t to the i n h i b i t o r y e f f e c t s of nonionic s u r f a c t a n t s on the a n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s through p r o t e c t i v e a c t i o n on micro-organisms, s e v e r a l authors have demonstrated t h a t n o n i o n i c s p o t e n t i a t e the a n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s . However, i n a d d i t i o n to the v a r i o u s f a c t o r s discussed i n the l i t e r a t u r e survey an apparent s y n e r g i s t i c e f f e c t above CMC could be merely an a r t i f a c t due to the use of an o v e r s i m p l i -f i e d physico-chemical model. In the present work Eq.28 was used to p r e d i c t the t o t a l c o n c e n t r a t i o n of p r e s e r v a t i v e r e q u i r e d i n the s u r f a c t a n t s o l u t i o n i n order to achieve the d e s i r e d c o n c e n t r a t i o n i n the aqueous phase. A good c o r r e l a t i o n between t h e o r e t i c a l l y generated p l o t s and experimentally d e t e r -mined values ( F i g . 35) showed that Eq.28 was v a l i d and adequately described the i n t e r a c t i o n . However, equation 61 has been used e x t e n s i v e l y f o r c a l c u l a t i n g the r e q u i r e d p r e s e r v a t i v e c o n c e n t r a t i o n i n s u r f a c t a n t s o l u t i o n s ( P a t e l and Kostenbauder, 1958; Pisano and Kostenbauder, 1959; Blaug and Ahsan, 1961a,b; Bahal and Kostenbauder, 1964; P a t e l and Foss, 1965; Ashworth and Heard,-1 9 6 6 ; P a t e l , 1967; Bean, Konning and Malcolm, 1969) [ D j — R = 1 + k[M] (Eq.61) [ D £ ] where k represents the b i n d i n g c a p a c i t y of the s u r f a c t a n t . For a given s u r f a c t a n t c o n c e n t r a t i o n the R v a l u e , or b i n d i n g or s o l u b i l i z a t i o n constant, 168 i s assumed to be independent of [ D £ ] , The t o t a l preservative concentration, [D ], i s calculated by multiplying the concentration of preservative required f o r antimicrobial action with the R values at an appropriate surfactant concentration, [ M ] . Equation 61 i s i n f a c t a s p e c i a l case of Eq.28 and, for a given macromolecule concentration, the R value w i l l be independent of [D £] only under two conditions: I. When [Df] — - — > 0 R = 1 + n 1 K 1 [ M ] + n 2 K 2 [ M ] (Eq.62a) or or R = 1 + k'[M] (Eq.62b) I I , When [D £] i s constant, as i n the case of saturated solutions ri K [ M ] n K [ M ] R = 1 + — _ i + . (Eq.63a) 1 + K^Dj] 1 + K 2[D f] R. = 1 + k"[M] (Eq.63b) Hence, k' (or k") does not f u l l y characterize the i n t e r a c t i o n . Table 8 shows a comparison between Eq.28 and 62 using the appropriate binding para-meters for the i n t e r a c t i o n of chlorocresol with cetomacrogol. The values of t o t a l preservative concentration [D^], calculated using Eq.62 are approxi-mately three times higher than the [D t] values obtained using Eq.28. Thus, i t i s not v a l i d to assume that R i s independent of [D^] i n p r a c t i c a l s i t u a t i o n s . An apparent synergism reported by Pisano and Kostenbauder (1959) 169-on the b a s i s of comparison between the MIC determined experimentally and the MIC c a l c u l a t e d using Eq.61 might be due to a higher c a l c u l a t e d value of t o t a l p r e s e r v a t i v e c o n c e n t r a t i o n than to r e a l synergism. TABLE 8. Comparison Between Equation 28 and 62 f o r the I n t e r a c t i o n of C h l o r o c r e s o l w i t h Cetomacrogol moles 1 x 1 0 3 T o t a l P r e s e r v a t i v e Concentration [DL]* [M] [D f] [D t] - D>;] r C a l c u l a t e d using i- C a l c u l a t e d using= t C a l c u l a t e d using= Eq.64 and [Dj] Eq.28 Eq.62 7.69 2.45 12.22 32.44 8.67 23.08 ti 31.76 92/42 10.35 38.46 II 51.'29 152.4 10.8 i s the c o n c e n t r a t i o n of f r e e p r e s e r v a t i v e c a l c u l a t e d f o r a given [D£], Table 8 shows that the values of f r e e p r e s e r v a t i v e c o n c e n t r a t i o n , 'E'Dj], corresponding to [D^], are approximately four times higher than the value of [D^] and, moreover, are not constant. Thus, i n the present work, an apparent synergism between p r e s e r v a t i v e and s u r f a c t a n t would have been observed i f the t o t a l p r e s e r v a t i v e c o n c e n t r a t i o n were c a l c u l a t e d using Eq.62. The values of [D^] were c a l c u l a t e d using Eq.64 which i s a rearrangement of Eq.28. The r o o t s of Eq.64 were c a l c u l a t e d u s i n g Bairstow's method (U.B.C. computer program, ZPOLY). 170 K1 K2^ D P 3 + [ K 1 + K 2 + n i K i K 2 M + n 2 K l K 2 M " K l K 2 ^ D t ^ ] ^ D f ^ 2 + [ l + njKjM + n 2K 2[M] - K^D^] - ^ [ V ] ] [D'] - [Dj] = 0 (Eq.64) (d) B a c t e r i c i d a l A c t i v i t y of C h l o r o c r e s o l i n L i q u i d P a r a f f i n Emulsions S t a b i l i z e d w i t h Cetomacrogol Figure 45 shows p r o b i t % s u r v i v o r s p l o t t e d as a f u n c t i o n of time f o r the b a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n water (curve A) and l i q u i d p a r a f f i n emulsions of v a r y i n g o i l - w a t e r r a t i o s (curves B, C and D) against E_. c o l i . The symbols represent experimental data, w h i l e the l i n e s are f i t t e d using the l e a s t squares method. The i n i t i a l v alue of [D^] i s the same f o r a l l the curves, although the t o t a l p r e s e r v a t i v e c o n c e n t r a t i o n , [ D ] , v a r i e s . The r e s u l t s of c o n t r o l experiments are shown i n F i g . 39(C). The i v i a b i l i t y of E. c o l i i n l i q u i d p a r a f f i n emulsions of v a r y i n g o i l - w a t e r r a t i o , q, remained almost unchanged during the 8 hour i n t e r v a l . A s t a t i s t i c a l a n a l y s i s of the slopes of the curves i n F i g . 45 was performed using a s i m i l a r method to that described i n the previous s e c t i o n f o r F i g . 42. The r e s u l t s are given i n Table 9. D i f f e r e n c e s between the slopes of the curves A and B, B and C, C- and D are n o n s i g n i f i c a n t . The d i f f e r e n c e between the slopes of the curve A and curves C and D i s s i g n i f i -cant. S i m i l a r l y there i s s i g n i f i c a n t d i f f e r e n c e between the slopes of curves B and D. Thus the d i f f e r e n c e between the slopes of any two adjacent curves i s n o n s i g n i f i c a n t . However, there i s a s i g n i f i c a n t d i f f e r e n c e between the slopes of a l t e r n a t e curves. This suggests that the magnitude of d e v i a t i o n of the slopes of curves B, C and D from the slope of curve A i s 171 1 1 1 I _L_ I I I 1. 2 3 4 5 6 7 8 HOURS • . 45. Probit % survivors of _E. c o l i as a function of time for the b a c t e r i -c i d a l a c t i v i t y of chlorocresol i n water (Curve A, from F i g . 41) and l i q u i d - p a r a f f i n emulsions s t a b i l i z e d with cetomacrogol. Oil-water r a t i o , q, of the emulsions: , 1.0; . C • , 0.5; DO , 0.2. Ceto-macrogol concentration = 3.0%. I n i t i a l free preservative concentra-ti o n , [D^], for water and the emulsions = 0.035%. Total preservative concentration, [D ], %: A, 0.035; B, 0.2557; C, 0.3214; D, 0.3871. Points experimental, l i n e s f i t t e d using least squares method. TABLE 9. Comparison of Slopes in Fig. 45 Using t Test Curve t Calculated using Eq.59 Modified Welch Degrees of Freedom (Eq.60) Probability Comparison of curves B, C and D with curve A B 1.67 28.14 < 0.1 C 3.83 31.33 <C 0.005 D 5.53 25.24 << 0.005 Comparison of. curves B and C with curve B G 1.28 30,02 < 0.2 D 2.11 24.37 <C 0.025 Comparison of curve C with curve D D 0.91 36.33 <0.2 173 s m a l l . Hence, a n t i m i c r o b i a l a c t i v i t y of c h l o r o c r e s o l i n l i q u i d p a r a f f i n emulsions i s l a r g e l y a f u n c t i o n of The small d i f f e r e n c e s between the slopes of curves B, C and D als o supports t h i s view. A s i g n i f i c a n t d i f f e r -ence between the curves B and D suggests that the o i l - w a t e r r a t i o does a f f e c t the a n t i m i c r o b i a l a c t i v i t y of the p r e s e r v a t i v e , and i n c r e a s i n g the o i l - w a t e r r a t i o r e s u l t s i n an increase i n the a n t i m i c r o b i a l a c t i v i t y . However, the e f f e c t i s of small magnitude. Recently Bean, Konning and Malcolm (1969) and P a t e l and Romanowski (1970) used mathematical models to p r e d i c t the r e q u i r e d p r e s e r v a t i v e concen-t r a t i o n and reported s a t i s f a c t o r y c o r r e l a t i o n s using m i c r o b i o l o g i c a l techniques. Some aspects of t h e i r work are open to c r i t i c i s m f o r the f o l l o w i n g reasons: I . The b i n d i n g parameters used to c h a r a c t e r i z e the i n t e r a c t i o n between p r e s e r v a t i v e and s u r f a c t a n t do not f u l l y d e s c r i b e the i n t e r a c t i o n . This aspect i s discussed i n the previous s e c t i o n ( c r i t i c i s m regarding Eq.61). i Furthermore P a t e l and Romanowski (1970) used cellophane membrane i n t h e i r study of the p r e s e r v a t i v e - s u r f a c t a n t i n t e r a c t i o n using the e q u i l i b r i u m d i a l y s i s technique. In an e a r l i e r p a r t of the d i s c u s s i o n (C) i t was shown that using cellophane membrane i n i n t e r a c t i o n s t u d i e s introduced appreciable e r r o r s i n t o the b i n d i n g parameters due to osmosis and permeation of the s u r f a c t a n t through the d i a l y s i s membrane. Thus, the v a l i d i t y of the bi n d i n g r e s u l t s used by P a t e l e_t a l . i n t h e i r mathematical model i s doubt-f u l . Bean et a l . (1969) a l s o used the e q u i l i b r i u m d i a l y s i s technique f o r studying p r e s e r v a t i v e - s u r f a c t a n t i n t e r a c t i o n . However, they d i d not rep o r t the type of d i a l y s i s membrane used. I I . The m i c r o b i o l o g i c a l experimental technique of P a t e l e_t a l . (1970) 174 i s open to c r i t i c i s m because the micro-organisms were not i n d i r e c t contact with the emulsion. The work of Bean et a l . (1962) on simple o i l -water dispersions suggests that antimicrobial a c t i v i t y i s not simply a function of the concentration of free preservative and that preservative at the oil-water i n t e r f a c e plays a part. However, the r e s u l t s of the present study show that t h i s e f f e c t i s of small magnitude. I I I . P a t e l and Romanowski (1970) compared minimum i n h i b i t o r y concentration (MIC) of a preservative i n an emulsified system with the concentration of preservative calculated using a mathematical model (Eq.44) . and a value of MIC determined i n a growth promoting medium. This assumes that the MIC determined i n a growth promoting medium p a r a l l e l s the MIC i n the aqueous phase of the emulsion. The choice of the MIC as the concentration of free preservative required i n the aqueous phase of the system, [D^], i s , of course, a rough approximation of the amount a c t u a l l y needed to preserve a complex disperse system. MIC values are normally determined i n a nutrie n t growth medium and depend on factors such as oxygen tension, i n t e r f a c i a l tension, osmotic pressure, degree of aeration, etc., as we l l as the n u t r i t i v e value of the medium. The n u t r i t i v e value of the aqueous phase of an emulsified system i s u n l i k e l y to betas great as that of nutrient broth or other growth media, although, as pointed out by Tenenbaum (1967), the n u t r i t i v e q u a l i t y of "cosmetic and pharmaceutical formulations often ranges from water solutions to high p r o t e i n soups" and i s frequently overlooked when' attempting to preserve a product. However, i n most cases the n u t r i t i v e value of the preparation w i l l be l e s s than that of the growth medium i n which the MIC was determined and the [Dj] value w i l l therefore exceed minimum concentration required to i n h i b i t growth. 175 IV. Bean et_ a l . (1969) used an e x t i n c t i o n time method ( l i t e r a t u r e survey, 1(b)2) f o r studying the a n t i m i c r o b i a l a c t i v i t y of p r e s e r v a t i v e s i n e m u l s i f i e d systems. There are a few weaknesses i n the e x t i n c t i o n time method when a p p l i e d to emulsion systems. For example, i t was d i f f i c u l t to d i s t i n g u i s h between the t u r b i d i t y due to the growth of micro-organisms from t u r b i d i t y due to o i l d r o p l e t s . However, the authors overcame t h i s problem by the use of i n d i c a t o r b r o t h . A more s e r i o u s disadvantage of t h i s method i s the carry-over of an a p p r e c i a b l e amount of p r e s e r v a t i v e , a s s o c i a t e d w i t h the o i l d r o p l e t s or the s u r f a c t a n t m i c e l l e s , to the growth medium. This r e s i d u a l p r e s e r v a t i v e can i n h i b i t the growth of micro-organisms. I n the present work the problem of carry-over was avoided by the use of the M i l l i p o r e f i l t r a t i o n technique. Bean, Richards and Thomas (1962) demonstrated that on d i s p e r s i n g l i q u i d p a r a f f i n i n an aqueous phenol s o l u t i o n greater a n t i m i c r o b i a l a c t i v i t y was observed than was observed without the o i l . I n v e s t i g a t i o n of t h i s system i n d i c a t e d that both micro-organisms and phenol were i n higher c o n c e n t r a t i o n at the i n t e r f a c e than i n the bulk aqueous phase and the increased a c t i v i t y of the d i s p e r s i o n was a t t r i b u t e d to t h i s i n t e r f a c i a l a d s o r p t i o n . Since the i n t e r f a c i a l area of o i l - w a t e r d i s p e r s i o n s s t a b i l i z e d w i t h e m u l s i f i e r s i s very much l a r g e r than simple o i l - w a t e r d i s p e r s i o n s , the i n t e r f a c i a l e f f e c t , on the a n t i m i c r o b i a l a c t i v i t y of a p r e s e r v a t i v e i n a s t a b i l i z e d o i l - w a t e r d i s p e r s i o n should be much more s i g n i f i c a n t than demon-s t r a t e d by Bean and others. F i g u r e 45 shows t h a t , although the d i f f e r e n c e between the slopes of curves B (q = 1.0) and D (q = 0.2) i s s i g n i f i c a n t , the magnitude of the d i f f e r e n c e i s s m a l l . Hence, the e f f e c t of o i l - w a t e r i n t e r -face and the c l o s e p r o x i m i t y between micro-organisms and p r e s e r v a t i v e r i c h 1 7 6 o i l droplets on the an t i m i c r o b i a l a c t i v i t y of preservatives i n emulsified systems i s of minor importance. Hence, a r e a l i s t i c test of the theory that preservative action depends on [D £] i s to compare the v i a b i l i t y of a given micro-organism i n emulsions with the same [D ] and varying [D] values. Equal b i o l o g i c a l response i n emulsions with d i f f e r i n g oil-water r a t i o s would then be an i n d i c a t i o n that the .antimicrobial a c t i v i t y i n emulsified systems i s mainly a function of [ D £ ] , and that the preservative i n the o i l phase or bound with surfactant micelles i s b i o l o g i c a l l y i n a c t i v e . This approach was used i n the design of the experiments i n the present work (Fig. 4 5 ) . In the previous section, the concept of capacity was developed f o r the surfactant systems. This concept w i l l now be extended to oil-water dispersions and emulsions (oil-water dispersions s t a b i l i z e d with a surf a c t a n t ) . ( i ) Oil-water dispersions: .Figure 4 6 shows a d i r e c t r e l a t i o n s h i p between the percent loss of chl o r o c r e s o l from water and the aqueous phase of hypothetical oil-water dispersions (see appendix 6 for the c a l c u l a t i o n of t h i s parameter) when an equal amount of preservative i s removed from water and the oil-water dispersion. For a given percent l o s s of preservative from water, the percent loss from the aqueous phase of oil-water dispersion i s a function of the oil-water p a r t i t i o n c o e f f i c i e n t (K°) and the oil-water w ' r a t i o (q). The percent loss from the aqueous phase of oil-water dispecsion .ia>o increases with an increase i n q when i s equal to 0.1; i t i s independent of q when K° i s equal to 1.0, and decreases with an increase i n q when K° i s n w n ' • ^ w equal to 10.0. As before, the capacity of a system to r e s i s t changes i n [D^] i s i n v e r s e l y proportional to the slope of a given l i n e . A slope equal to or 177 H' G' F.' E' D' ' C' B' A' X Loss of C h l o r o c r e s o l From Water (%) F i g . 46. Comparison between percent l o s s of c h l o r o c r e s o l from water and aqueous phase of an h y p o t h e t i c a l o i l - x r a t e r d i s p e r s i o n when equal amounts of p r e s e r v a t i v e are removed from water and the 0/W d i s p e r s i o n . K° = 0.1; o i l - w a t e r r a t i o , q: A', 0.1; w B', 0.2; C , 0.4; D', 0.6; E 1, 1.0; F', 2.0; G\ 3.0; H', 5.0;' K° = .1.0; q: X, 0.0 to oo. K° = 10.0; q: A, 0.1; B, 0.2; . w w C, 0.4; D, 0.6; E, 1.0; F, 5.0. 178 greater than one means that the system o f f e r s no resistance to changes i n [ D f ] . Figure 47 shows a d i r e c t r e l a t i o n s h i p between capacity (the r e c i p r o c a l of the slope of a given l i n e i n F i g . 46) and K° for various w values of q. For a given q, the capacity increases when K° i s more than w 1.0 and decreases when K° i s l e s s than 1.0. When K° i s equal to 1.0, the w w ' capacity i s independent of q (see F i g . 48) and i t s value.'is unity (the capacity. ,of water i s also equal to unity) . Figure 48 shows a c u r v i l i n e a r r e l a t i o n s h i p between capacity and q f o r various values of K°. The capacity increases with an increase i n q when K° i s more than 1.0. The capacity decreases with an increase i n q when K° i s l e s s than 1.0. The capacity i s independent of q when K° i s equal to 1.0. Hence, from F i g s . 47 and 48 i t i s apparent that both K° and q co n t r o l .the capacity of an oil-water dispersion. ( i i ) Emulsions: Figure 49 shows a d i r e c t r e l a t i o n s h i p between the percent loss of chlorocresol from water and the aqueous phase of hypothetical emulsions (see appendix 7 for the c a l c u l a t i o n of t h i s parameter) when an equal amount of preservative i s removed from water and the emulsion. For a given percent l o s s of preservative from water, the percent loss from the aqueous phase of the emulsion i s a function of q, K° and [M]. As before, w the capacity of a system to r e s i s t changes i n [D^] i s inversely proportional to the slope of a given l i n e . A slope equal to or greater than one means that the system o f f e r s no resistance to changes i n [D^] . For a given o i l -water r a t i o the value of slope i s a function of [M] and K°. Figure 49 shows that f o r a given [M] an increase i n q r e s u l t s i n a decrease i n the value of 179 Fig. 47. Capacity as a function of oil-water partition coefficient, K , for w the distribution of chlorocresol in hypothetical oil-water disper-sions. Oil-water ratio: A, 0.1; B, 0.2; C, 0.4; D, 0.6; E, 0.8; F, 1.0; G, 2.0; H, 3.0; I, 4.0; J, 5.0. I I— I _ l _ _ L I 0 1 2 3 . 4 5 1 • F i g . 48. Capacity as a f u n c t i o n of o i l - w a t e r r a t i o , q, f o r the d i s t r i b u t i o n of c h l o r o c r e s o l i n h y p o t h e t i c a l o i l - w a t e r d i s p e r s i o n s . . K°: A, 0.1; B, 0.5; C, 1.0; I),'3.0; w E, 5.0; F, 7.0; G. 10.0. 181 50 B' 40 30 20 10 V ' 1 I / / /•.'••#'• I f • 10 20 30 40 Loss of C h l o r o c r e s o l From Water (%) X 3 "2 A' X, 50 Comparison between percent l o s s of c h l o r o c r e s o l from water and aqueous phase of an h y p o t h e t i c a l o i l - w a t e r emulsion s t a b i -l i z e d w i t h cetomacrogol when equal amount of p r e s e r v a t i v e are removed from water and the emulsion. K° = 0.1; q: A', 0.1; B', 1.0; C, 5.0. K° = 1.0; q: X., 0.1; X., 1.0; X 0, 5.0. w I Z j K° = 10.0; q: A, 0.1; B, 0.2; C, 0.4; D, 0.6; E, 1.0; F, 5.0. Cetomacrogol concentration = 0.1%. 182 the slope when K° is equal to 10.0. For the same [M], increasing q results in an increase in the value of the slope when K° is equal to 0.1 and 1.0. Thus, unlike surfactant and oil-water systems where capacity i s determined only by [Mt]' and K°, q, respectively, in emulsified systems the w capacity is a resultant of the interaction between the parameters [M], K° and q. Figure 50 shows a curvilinear relationship between capacity w (the reciprocal of the slope of a given line in Fig. 49) and q for various K° at a constant value of [M], The intercept gives the capacity of the aqueous phase (containing surfactant) i n the absence of the o i l phase. At a fixed [M], increasing q decreases the capacity up to a certain value of K° (curves A, B and C), beyond that value an increase in q results in an increase in capacity (curves D, E and F). This i s because in emulsified systems the overall capacity of the system is determined by two factors -the capacity of the o i l phase and the capacity of the aqueous phase containing the surfactant. The capacity of the o i l phase is governed by K° while the capacity of the aqueous phase i s controlled by the surfactant w concentration. If the capacity of the o i l i s less than the capacity of the aqueous surfactant phase, then an increase in q w i l l result in a decrease in the overall capacity of the system (curves A, B and C). This aspect i s ' further illustrated in Fig. 51 for the liquid paraffin emulsions used in this study. Conversely, i f the capacity of the aqueous phase is less than the capacity of the o i l phase, then an increase in q w i l l result in an increase in the capacity (curves D, Eand F). As the capacity of the aqueous phase approaches the capacity of water, e.g., with very dilute surfactant solutions, an increase i n q w i l l result in an increase in the * o capacity for a l l values of K greater than 1.0 (compare Figs. 48, 50 and 52). I L 1 1 I I 0 1 . 2 3 4 5 q F i g . 50. Capacity as a f u n c t i o n of o i l - w a t e r r a t i o , q, f o r the d i s t r i b u t i o n of. c h l o r o c r e s o l i n h y p o t h e t i c a l 0/W emulsions s t a b i l i z e d w i t h cetomacrogol. Cetomacrogol con c e n t r a t i o n = 1.0%. K°: A, 0.1; B, 1.0; C, 3.0; D, 5.0; E, 7.0; w F, 10.0. 184 9 -H M O 5! F i g . 51. Capacity as a f u n c t i o n of o i l - w a t e r r a t i o , q, f o r the d i s t r i b u t i o n of c h l o r o c r e s o l i n l i q u i d - p a r a f f i n emulsions s t a b i l i z e d x j i t h cetomacrogol. Cetomacrgol concentration = 3.0%. K°, l i q u i d - p a r a f f i n = 1.67. 185 0 1 2 3 4 5 q F i g . 52. Capacity as a f u n c t i o n of o i l - w a t e r r a t i o , q, f o r the d i s t r i b u t i o n of c h l o r o c r e s o l i n h y p o t h e t i c a l 0/W emulsions s t a b i l i z e d w i t h cetomacrogol. Cetomacrogol concentration = 0.1%. K°: A, 0.1; B, 1.0; C, 3.0; w D, 5.0; E, 7.0; F, 10.0. 186 The capacity of a system c l e a r l y a f f e c t s i t s a b i l i t y to withstand microbial contamination. High capacity systems are able to r e s i s t losses i n the concentration of preservative due to such factors as: I. adsorption onto or complexation with the container and closure surfaces I I . adsorption, absorption of chemical reaction with contaminants including micro-organisms •III. chemical decomposition. I t i s apparent that the o v e r a l l effectiveness of a preservative i n a complex disperse system w i l l depend on the capacity of the system i n addition to the concentration of free preservative. The magnitude of the capacity f a c t o r s ' contribution to the apparent o v e r a l l a n t i m i c r o b i a l a c t i v i t y of a preservative w i l l vary according to the t e s t i n g procedure adopted. Thus, the e f f e c t of capacity i s more l i k e l y to become apparent i n procedures which involve sampling f o r micro-organisms over prolonged time periods, e.g., days, weeks or months compared with hours i n the present work, and challenge tests i n which the preparation i s repeatedly inoculated with micro-organisms over a long storage period. Such procedures allow time for the preservative to r e - e q u i l i b r a t e between the various phases following any depletion and for t h i s r e - e q u i l i b r a t i o n to exert i t s e f f e c t on the a n t i m i c r o b i a l action. The object of the present work was to test the hypothesis that a n t i m i c r o b i a l a c t i v i t y depends mainly on the concentration of free preservative i n the aqueous phase and to evaluate the e f f e c t of factors such as changes i n surfactant concentration and oil-water r a t i o . The experimental conditions chosen were such that capacity factors are u n l i k e l y to have a s i g n i f i c a n t e f f e c t on the r e s u l t s . However, incomplete understanding of the capacity f a c t o r and i t s s i g n i f i c a n c e i s a probable reason for much of the controversy 187 i n the l i t e r a t u r e regarding the evaluation of preservative effectiveness i n s o l u b i l i z e d and emulsified systems. The concept of capacity, as i l l u s t r a t e d q u a n t i t a t i v e l y i n t h i s work, not only emphasizes i t s importance i n the design of experiments for the evaluation of t h e o r e t i c a l models, but i t can be used i n the formulation of e f f e c t i v e preservative systems. The graphical methods used f or r e l a t i n g the capacity and the various physicochemical parameters, such as K ° , [ M ] w and q, can help i n s e l e c t i n g an appropriate preservative which w i l l provide high capacity without conferring undue t o x i c i t y to the given formulation. In view of the number of v a r i a b l e s involved i n the preservation of s o l u b i l i z e d and emulsified systems, i t i s r e a l i z e d that physico-chemical methods cannot replace a f i n a l m i c r o b i o l o g i c a l evaluation of the product for i t s a b i l i t y to withstand microbial contamination. However, physico-chemical methods do provide a l o g i c a l f i r s t step i n estimating the required preservative concentration, and thus help i n avoiding much ' t r i a l and error' formulation which i s common where purely m i c r o b i o l o g i c a l techniques are employed. 188 SUMMARY 1. Permeability of Membrane to Nonionic Surfactants The permeability of two brands of cellophane membranga'•-arid, a s i l i c o n e membrane to a nonionic surfactant, cetomacrogol, was investigated using equilibrium d i a l y s i s , dynamic d i a l y s i s , and an u l t r a f i l t r a t i o n tech-nique. The permeability of Fisher cellophane membrane to three n - a l k y l polyoxyethylene surfactants was tested using the equilibrium d i a l y s i s technique. The r e s u l t s show that cellophane membranes are permeable to the nonionic surfactants, while s i l i c o n e membrane i s impermeable to the same surfactants. 2. Osmosis i n D i a l y s i s Studies Involving Nonionic Surfactants Equilibrium and dynamic d i a l y s i s studies showed increase i n the volume of the surfactant s o l u t i o n due to osmosis when cellophane was used as a semipermeable membrane. No osmotic e f f e c t was observed using s i l i c o n e membranes. 3. Interaction of Preservatives with Nonionic Surfactants (a) Equilibrium d i a l y s i s Cellophane and s i l i c o n e membranes were compared i n an equilibrium d i a l y s i s study of the i n t e r a c t i o n of chlorocresol with n - a l k y l polyoxyethylene surfactants. Appreciable errors were introduced into the binding parameters when cellophane was used as the d i a l y s i s membrane. These errors are due both to osmosis and to permeability of the membrane to the surfactants, with d i l u t i o n of surfactant as a r e s u l t of the osmotic d i f f e r e n t i a l across the membrane being the major f a c t o r . I t i s suggested that the use of cellophane 189 as a membrane can introduce appreciable errors into i n t e r a c t i o n studies in v o l v i n g nonionic surfactants unless corrections are made for osmosis. S i l i c o n e rubber i s a s a t i s f a c t o r y membrane for these studies. (b) D i a f i l t r a t i o n technique The d i a f i l t r a t i o n technique was compared with equilibrium d i a l y s i s f or the i n t e r a c t i o n of benzoic a c i d with cetomacrogol. The r e s u l t s were expressed i n the form of Scatchard p l o t . When the equilibrium d i a l y s i s technique was used, binding of the benzoic a c i d with cetomacrogol was independent of the surfactant concentration. However, the r e s u l t s obtained using the d i a f i l t r a t i o n technique showed a dependence of the binding curves on the surfactant concentration. This anomalous behavior i s a t t r i b u t e d to various technical a r t i f a c t s of the method and not to changes i n the c h a r a c t e r i s t i c s of the surfactant molecules during d i a f i l t r a t i o n . 4. Interaction of Preservative Mixtures with Cetomacrogol Interaction of various binary preservative mixtures with cetomacro-gol was studied using the equilibrium d i a l y s i s technique. The binding of a preservative with cetomacrogol was a l t e r e d i n the presence of another preservative. This observation i s s i m i l a r to the phenomena of competitive binding of.drugs with macromolecules. However, no c o r r e l a t i o n was found between the binding behavior of preservative mixtures predicted using the theory of competitive binding and the observed values. The disagreement between the predicted values and the experimental r e s u l t s i s explained i n terms of the locus of s o l u b i l i z a t i o n of preservatives i n a surfactant m i c e l l e , and changes i n the number as well as behavior of binding s i t e s i n a surfactant m i c e l l e upon i n t e r a c t i o n with the preservatives. 190 5. I n t e r a c t i o n of C h l o r o c r e s o l w i t h Mixtures of Some Nonionic Surfactants The i n t e r a c t i o n of c h l o r o c r e s o l w i t h b i n a r y mixtures of nonionic s u r f a c t a n t s was studi e d using the e q u i l i b r i u m d i a l y s i s technique. An attempt was made to p r e d i c t the b i n d i n g behavior of s u r f a c t a n t mixtures u s i n g b i n d i n g parameters which c h a r a c t e r i z e the i n t e r a c t i o n of a p r e s e r v a t i v e w i t h i n d i v i d u a l s u r f a c t a n t s . Good agreement was found between the p r e d i c t e d and the experimental v a l u e s . In a d d i t i o n i t was shown that b i n d i n g i s a d i r e c t f u n c t i o n of the HLB value of nonionic s u r f a c t a n t s . Since HLB i s an a d d i t i v e property, the b i n d i n g of a p r e s e r v a t i v e w i t h a s u r f a c t a n t mixture i s an a d d i t i v e f u n c t i o n of the bi n d i n g of the p r e s e r v a t i v e w i t h the i n d i v i d u a l s u r f a c t a n t s . 6. D i s t r i b u t i o n of C h l o r o c r e s o l i n L i q u i d Paraffin-Water Systems The d i s t r i b u t i o n of c h l o r o c r e s o l between l i q u i d p a r a f f i n and water was s t u d i e d using a shake-out method. The d i s t r i b u t i o n obeyed a simple p a r t i t i o n law. 7. C a l c u l a t i o n of T o t a l Concentration of C h l o r o c r e s o l Required i n Cetomacrogol S o l u t i o n s to Produce the Desired Concentration of Free P r e s e r v a t i v e i n the Aqueous Phase The v a l i d i t y of Eq.28 was checked usi n g the e q u i l i b r i u m d i a l y s i s technique. Good c o r r e l a t i o n was found between p r e d i c t e d and experimentally determined v a l u e s . 8. C a l c u l a t i o n of T o t a l Concentration of C h l o r o c r e s o l Required i n L i q u i d P a r a f f i n Emulsions to Produce the Desired Concentration of Free P r e s e r v a t i v e i n the Aqueous Phase 191 The v a l i d i t y of Eq.48 was checked for the d i s t r i b u t i o n of chlorocresol between l i q u i d paraffin-water-cetomacrogol systems using the equilibrium d i a l y s i s technique. Good c o r r e l a t i o n was found between predicted.and experimentally determined values. 9. C o r r e l a t i o n of Physico-chemical Data with Antimicrobial A c t i v i t y (a) Comparison between M i l l i p o r e and pour-plate techniques The M i l l i p o r e f i l t r a t i o n method was compared with a pour-plate technique for the v i a b l e counting of E. c o l i . Good c o r r e l a t i o n was found between the two techniques. (b) B a c t e r i c i d a l a c t i v i t y of c h l o r o cresol i n water The b a c t e r i c i d a l a c t i v i t y of c h l o r o cresol i n water against E. c o l i was studied using a v i a b l e count method. The r e s u l t s were expressed as log number of organisms surviving, or p r o b i t % survivors as a function of time. Graphical presentation of the data using probit ulots i s recommended because the l a t t e r give s t r a i g h t l i n e r e l a t i o n s h i p s . The b a c t e r i c i d a l a c t i v i t y of c h l o r o c r eso l i n water against E_. c o l i i s a function of the concentration of the preservative. Increasing the concentration of chlorocresol from 0.02% to 0.03% makes l i t t l e change i n the death rate of E_. c o l i . ' However, a concentration of 0.035% c h l o r o c resol produced 99% m o r t a l i t y within 6-8 hours. (c) B a c t e r i c i d a l a c t i v i t y of chlorocresol i n aqueous solutions of cetomacrogol The b a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n various concentrations of cetomacrogol against E_. c o l i was studied using the v i a b l e count method. 192 S t a t i s t i c a l analysis of the probit p l o t of the data indicated that the b a c t e r i c i d a l a c t i v i t y of chlorocresol i n cetomacrogol solutions was l a r g e l y a function of the free preservative concentration i n the aqueous phase. However, an increase i n cetomacrogol concentration produced a s i g n i f i c a n t but small decrease i n a n t i b a c t e r i a l a c t i v i t y . (d) B a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n l i q u i d p a r a f f i n emulsions s t a b i l i z e d with cetomacrogol The b a c t e r i c i d a l a c t i v i t y of c h l o r o c r e s o l i n l i q u i d p a r a f f i n emulsions of varying oil-water r a t i o s against E_. c o l i was studied using the v i a b l e count method. S t a t i s t i c a l analysis of the probit p l o t of the data indicated that the a n t i b a c t e r i a l a c t i v i t y of chlorocresol i n l i q u i d p a r a f f i n emulsions was l a r g e l y a function of the concentration of f r e e preservative i n the aqueous phase. A s i g n i f i c a n t but small increase i n the a n t i b a c t e r i a l a c t i v i t y with increase i n the oil-water r a t i o i s possibly due to i n t e r f a c i a l e f f e c t s . 193 REFERENCES Alexander, A.E. and Trim, A.R. (1946). Proc, Roy. S o c , B113, 220-234, Alexander, A.E. and Tomlison, A.J..-H. (1949). 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Pharmac, _T7, 22S.-27S. Withington, R. and C o l l e t t , J.H. (1972). i b i d . , 24, Suppl. 131P. 205 Withington, R. and C o l l e t t , J.H. (1973). i b i d . , 25, 273-280. WolfhUgel, G. and von Knorre, G. (1881). M i t t . K a i s e r l . Gesundheitsamt, 1, 352. Wolf, P.A. and Westveer, W.M. (1950). Arch. Biochem., 28, 201. APPENDICES 1. Sample Calculations for Estimating [D,] and [D^] Employing the D i a f i l t r a t i o n Technique Using the Data for the Inter-action of Benzoic Acid (BA) with Cetomacrogol (Fig.19) Volume Dilut i o n factor Absorbance 230 nm Concentration of BA.In the f i l t r a t e [ D f ] g . l . " 1 x 10 2 g. x 10 4 Concentration of BA bound . IOOO = Ab V m g . l . - 1 x 10 2 of f i l t r a t e V f ml. Amount of BA i n the f i l t r a t e = [ D J . V f Cumulative amount of BA i n the f i l t r a t e Amount of BA free i n the c e l l = TD 1 m L f J1000 = A' f Amount of BA enter-ing the c e l l = ^ 1 0 0 0 = A t Cumulative amount of BA entering the c e l l Amount of BA bound =Ev Af 1000 =Af E A f E A t _ E A f = \ . 4.5 5 0.82 4.5 2.02 2.02 22.5 41.98 41.98 17.46 3.49 4.5 10 0.672 7.3 3.28. •5.31 36.5 41.98 83.96 42.15 8.43 4.65 25 0.431 11.7 5.44 10.75 58.5 43.38 127.34 58.1 11.62 4.4 50 0.298 16.2 7.13 17.88 81.0 41.05 168.39 69.51 13.90 4.5 50 0.378 20.6 9.27 27.15 103.0 41.98 210.37 80.22 16.04 Volume of cetomacrogol in the c e l l , V = 50 ml. Concentration of benzoic acid i n the reservoir = 6.933 g . l . Absorptivity of benzoic acid i n 0.01N HCl at 230 nm. = 91.76. Concentration of cetomacrogol = 1.0%. ro o CTS 207 2. Calculation of the Binding Parameters for the Interaction of a Preservative with a Nonionic Surfactant , (a) For a given [M] , determine experimentally [D^] and [D £] for various values of [D t]. (b) Calculate r = D>b]/[M]. (c) Calculate values of n's and K's by f i t t i n g the values of r and [D £], with the aid of a nonlinear regression program (Meyer and Guttman,. 1968), to the Eq.25 n K [D ] n K [D ] r = 1 + — i — (Eq.25) 1 + K j [ D f ] 1 + K 2 [ D f ] (d) The regression program requires experimental values of r and [D £], and a rough estimate of the values of n's and K's. The more accurate these estimates are, the fewer the iterations required for the computer calculation. (e) The parameters calculated by the program include: fit t e d values of n^, K^ , n 2 and K2, fitt e d values of r <§3'r) for a given [D £], andCr/[D £], (f) Table 10 gives a l i s t i n g of the computer program and a sample of the results computed using the data for the interaction of chlorocresol with cetomacrogol (Fig. 17, silicone rubber data). oo o CN FC?TRA\ IV G CC N F 1L E R r'A IK C4-0fc-')4 14:55:46 PAGE 0001 C C 01 0002 CCPycK CV.X1 ,X2 ,Y .RZERCYMT CIMENSION X H 15C) , X2( 150) ,Y( 15C) ,DV( 1C) ,A(10) , SXX< 10 ,10) ,SXY(1 10),DIAG(10),CA(10),S(10,10),RDF<15C) • A2.a3.A--. , X) = Al*f2«X/(l.*Al*X) + /i3*A4*X/(l.+A3»X) > 0004 CCC5 0006 M = 4 PE AC (5 ,S02 ) N , I A ( I ) ,1 = 1,C) KP.I T E ( £ ,SC2» N , ( A l l ) , 1 = 1 , M COO 7 902 FCPKA1(13 ,4615.8) CCCE 0 009 DC 5C K = l,rv R.EADI 5 ,SCC) Y ( K ) , X 1 ( K ) CC1C C011 CO 1 2 <;oo "5C4 FLPCAT 12F10.0) F0RPAT(E11.5,EE.l) W F . I T E U , S C 4 ) Y ( K ) , X 1 ( K I : CC13 5C CCMIM'E 0014 0015 HACP=1. V = 10 . CC16 c a n CC I 8 C = l . E - 2 TEST = 'l.L-4 ITER=0 C O S C020 CC21 70 • DC 7C 1 = 1,1' CA(I)=C. C E M IMJE , TABLE 10. Computer Program For The C022 0023 002 4 ICC S S E = S S C E ( A , [ : A , N , ! V ) I T E R . = I T E R + 1 . C A L L S O R S ( N , S X X , S X Y , A , C I A G , M ) Nonlinear Regression Analysis of the CC25 . 0026 C C2 7 KPI1=1 T C = C / V I F ( C - 1 . E - a ) 1 2 1 , 1 2 1 , 1 1 0 Binding Data. C028 110 CALL SLVMSXX , S X Y , 1C , C I AG ,CA ,S ,M 002 9 CC3C 116 T S S E = S S C E ( A , C A , N , M ) IF ( T S S E - S S E ) 2 0 0 , 1 3 0 , 120 003 1 13C 1F( TC-i.ErE)12C,3CC,30C 0032 CC23 12C 121 k R IT = K R I T + 1 1 C = T C * V 0024 GO TO 11C 0035 CC3fc 200 C = TC S S E = USC 0037 CC33 C C 3 S Kt!CL = l C C 220 1 = 1 , P A ( I ) = A ( I ) + CA( I) *CAKF 0040 CC41 CC42 2 1C QULU = ABS(DA( I ) )/( 1 . E - 3 + ABSIAII ) ) ) IF(CUCT-TEST )i2C,22C,210 K C C L = 2 U043 C044 004 5 22C 3CC CCNt U-iUE CX TO ( 3 C C , 1 0 0 ) , K C C L t-.F ITfcl t, 5CC1AI2) , A (1) , A ( 4 ) , A ( 3 ) 0046 500 FriRw.AT(12hCUEFFICIEKTS/(2E15.8)) CC47 C 0 A 8 5C 5 r R I T f ( 6 , 5 0 5 ) S S E F0R>1AT( 11HSSQ CF DEV. ,E15.£//6X,'DF ' , 15X ,1 R 1 ,12X, ,CR' ,12X, • R/DF 1 , 112X, ' C I F ' I CC4-; 03'JO C C 5 I =1 ,h CY = CALCY(A( 1 ) , A ( 2 ) , A ( 3 ) , A ( 4 ) , X 1 ( I ) ) CC5L CC52 0053 5 C I F = Y( I )-CY RDF1 I)=CY/X1 I 1) W P I T F 1 6 , 5 1 C ) X 1 1 I ) , Y U ) , C Y ,RDF(I) ,CIF CC54 510 FCRNAT<4E15.8,2X,E15.8) CC55 END 209 r\j ro o o a © n o o o o o n o| M M R -N) r - o vtf m —j! 7* X 7? -" C J C n o (/i n r. >rj <Z ZP- X o o o O r i O — Ct O O «T X j o o O o o o O C J o - J a- ml — o . n o c r> n r. o o o r> o o J> IjJ N ! O O l/ i : r » ~ f -r s: 33 • T l X n n ^ n 7 w c , —i n n -' < 2 m v CO X c — T I— CO o — 210 O O O C O O o o o o o n H- r- »- ("> C W N : i - O O U J — ^ o — I I n r*> r* n — r— 5» ~ o p- — C O C I I —> rr, d O O j o r> o r> tn <y o — — 2 C — O O O O o o r ; x - i u r o i— I I I I n -i - I- z 7 IV G COUP IL ER •SCE 14 :55 :47 PAGE 0001 0001 CCC2 0003 CCO* G005 00<J6 CCC7 CU03 o o o s CC 1C o c u CC12 CC13 FUI-CTICI^ SSCF(A,CA,/V,M) DINENSICN * (10 ) ,CAUC) ,TA(10) CINENSIPN X1<15C),X2(1EC),Y(15C),DW10) CCH"CN nv,Xl,X2,Y,f<ZERC,YNIT CALCY(A1,A2,A3,A4,X) CO IC 1=1,M TA ( I ) = A( I MCA ( I) = Al*A2*X/< l.+Al*X ) + A3*A4*X/ U.+A3*X) 1G CONTINLE SSCL' = 0 . CL 2C K = 1, FY=Y(k)-CALCY< T A ( 1 ) , T A ( 2 ) , T A < 3 ) , T A K ) , X 1 < K ) ) SSCE=SSCE+FY*FY C C k T I M E 001* CG15 PFTUPN FNC TOTAL MEMORY PF.CLIREMt.NTS CCC33E BYTES CC^FILE TIMF = C . l SECONCS FCPrRAN IV G CCrPILEF. f V FY CCC1 0002 0003 CCC* C005 CCCfc C C J 7 0CC3 CCCS 0010 C011 CC12 04-06-7* T*T577vF PAGE 0 0 0 1 S LFRCLT I fv E OVFY ( K , A , FY , l» J DIMENSION D V I 1 C I , A ( 1 C ) , X I I I 5 C ) , >2(15C),Y(150) CCfMIN nv,Xl,X2,Y,R2EBG,YlMT " ' L " ! - l 2 ' / 3 ' A * ' X > a * l * * * » X / 11 .-.Al*XHA3*A4*X/< l.-»A2*X) 1=1.M.2 TEPf = l . + A( I )*X1(K) »IUMEBMta||MI*>llKI-MI)»Alun<.>M«)»M(n t v i i ) = cvrn7TTrW *TFPMl '• r:V( I + l ) =A( I) *X1 (K) /1 ER ^  FY = V(K)-CALCY(A(1) , A ( 2 ) , A I 3 ) , A ( * 1 , >1(K)) RETURN FNO TCTAL MENCFY FFCUlRtMtNTS 0CC22A BYTES" ocyFIL£ TIWE = d stCCI^ CS ( FORTRAN IV G CCf-PILER C8 04-06-74 14:55:48 PAGE 0001 0001 SIBROL'TINE C R 1 A ,N , B ,K , OE 1) 000 2' C I iVEN'S ION Al 10 , 10) ,e< 1C ), IPVOT (1C ) , I NDEX I 10, 2 1 ,PIVCT(10) CCC3 ECLIVALEr>CbllRCr.,JRCW),<lCCL,JCCl) COU4 57 nei=i. J ? CC05 CCCfc CC07 17 LC 17 J=1,N IFVCT(J)=0 CO 12 5 I=1,N CCC8 T = 0. CCCS D G S J = l ,N 0010 I F ( I P V 0 T 1 J ) - 1 ) 1 3 , S , 1 3 001 1 13 CO 2 3 K=1,N CC12 I F ( I F V C T ( K ) - 1 ) 4 3 , 2 3 , 8 1 CO 13 43 IFIAeSll)-ABS(A(J,K))) 62, 23,23 C014 83 IK.Ch = J CC15 ICCL=K CClfc T=A(J,K) CC17 2 3 CCNT I M E CC18 S CCNT1MF CC19 IPV0T(IC0L)=IPVCT(ICCL)+1 CC2C IF( 1PCK- ICCL ) 73,10S,73 CC21 72 OET=-CET 0022 DC 12 L=1,N C023 T = A( IRCS-.L) CC24 A l l PCV> ,L ) =A(ICCL.L) 0025 ' 12 A( I C C L , L ) = T C026 IF if) ICS,109,33 0027 T=H( I ROr.) 0028 £ ( IREW )=El ICCL ) CC2S E( ICCL ) = T • C020 ICS INDEX! I,I)=IRUk CO3 L If.'CEXI l,2)=IC0L CC32 P]VCT( I ) = A<ICC I,ICCL) 0033 CFT=DET«PIVOTII) C034 A ( I C C L , I C C L ) = 1 . CC35 DC 2C5 L = 1 »N 0036 205 A( ICCL.L ) = A ( I C C L . L ) / P I V O T l I ) CO1 7 I F I f 1347 ,347,52 " " d o M C 0 3 9 CC4C 5J; 3^7 6(lCOU) = 9l lCO'U")/Pt V O T ( l ) BO 1:5 L I s l r N I F I L I - I C C L 1 2 1 ,135,21 004 1 21 T = A(L I , ICCL ) 0042 A(LI , ICCL1 = 0 . CC43 CC as L = 1,N 0044 es AIL I , L ) = A ( L I ,L ) - A ( I C C L , L ) * 1 0045 If ( M 135 ,135, 66 CC46 IB B(LI)=P 1 L I ) - B I I C C L ) * T 0047 125 CCiNT INUF CC48 222 CC 3 1=1,N CC4S L=N-I+1 0050 I F ( I N D E X I L , 1 ) - I N D E X ( L , 2 ) ) 1 S , 3 , 1 S C051 19 JRCVv = INCEX ( L , l ) C052 JCCL=INCEX(L,2) 0053 CC 54S K=1,N CC54 T=A(K,JPCW) CC55 A< K , JRCM =A ( K , JCCL) C056 A(K,JCOL) = T CC57 54S CCMIM'E V CC5E 3 C C M I M E : ; ; J 214 V A m Xi ml — i CN f ~ —' — — _ __ • EXECUTICN TERM INAT EC V tRUN -LOAD* 5=*S0URCE* f r Y r r i • T T n* • r r r r • i - " — \ / t A t l . U i 1 tl'\ C L t I N i 1 —————•--————______-_______-_, -26 C.54159985E 03 0.21599998E 01 0.91599991E 02 0.65999994E 01 1 DI l i . ' i : n 1 n / r r-~ ——— — — — : . • i c i o u r u i I J « — i • — _—— •1662UE 01 0.4E-02 • 155 7CE C l C.3E-C2 1 E C _ ,1 T r< 1 n —. r- r-. _ ' • ••• • .1 ,T AIT L.I 1..-" 1 1 • •15360E C l C.3E-C2 .1522CE C l C.3E-C2 1 C 1*1 1 f 1 r .1 - i r- r. — — • I D U t U t 01 U . i r — Us ~— — — -—— .1463QE C l 0.3E-C2 .14480E 01 0.3E-C2 1 i c l (i r n i "r> ~~r' r, — ' "1 — — ——_— -• 1 JO l u r (11 I! - - F- - fl f — _____ .1277CE C l C.2E-C2 .1229CE 01 0.2E-C2 i '  r ' - 7T~i ~ ~ r — " •' • 11 y t u : _i u . _ f L - U t _ ^ ™ — — — — — — - 1C.SCE C l C-2E-C2 •992CUE 00 0.2E-02 * " J C - I O L UU 0 . 7 h - 0 / ___—_____ .884CCE CC C. 1E-C2 •851G0E 00 0. 16-02 . C r "! i" r t- i~ — - i —• - -. — — • c*• 111fc _L U.lfc-C^ ______ _ ___ .837CCE CC 0.1E-C2 j ~~ — — •833CCE CC 0.1E-02 ' •tttCCt CC C.£F-C ? — — — ... .58_>CCE 00 0.7E-C2 •553CCE OC 0.6E-C3 ' 1 i r\ f r- n ? r- r- . — — — _ — _ — _ — _ . _ _ _ _ _ _ _ _ _ _ . • H O U u t U.*:t-L^ — _ .421006 00 0.5E-03 — , COEFFICIENTS 0.69673121E 00 C.1E69759CE C4 C. 13 352 E67E 03 0.21404743E 01 SSO OF CEV. 0.81691!9EE-C2 Of R CR CR/DF OIF 0.4C 169987E-C2 C.1E179993C 01 0 . l 7 7 l l l 6 - 3 E 01 C . 4 344 1 £ 5C E C3 ' 0 .46883583E-01 0.37199999F-02 G.16619997E C l C. 16629E24E C l C. 44 73C£EEE C3 -C.19826889E-02 0 .33519999E-02 0.15569992F 0 1 0 .1 5 5 20 85 9 F 01 C.46303271F C3 . 0.49 13 3 30 IE-02 C. 3"? EC <f< '9 9 E- C 2 C." 1 5 5 4 9T94T C l L : l T 6 T T£6~5T 01 C.4 616T?7T4~r. 03 . S^ T T^OTE^OT 0.34139999E-02 0.15279992E C l 0.1571C 5 7 2F C l C.46C1EC66F C3 -C. 3305 £ 1 £7E-01 0. 321 6999EE-02 0. 15219994E C l C.15105877E 01 0.46956.296F C2 0 . 11 4 11£ 6 7E-0 1 0.3201999SE-02 0. 15C19999E C l C.15C59£C5F 01 C.47031860F 03 -0.39606094E-02 C.31059999E-02 0.14679995E C l 0.14762592E 01 C.475292T2E C3 -C.82597733E-C2 Al 1LCa— V1 C Z 0 2__ t l ^ J l 1S CCCE C l 0.144ft0936C 01 G.48058936 F 03 0. 190£3_95CE-02 O.'2b72cCC'C"F.-0"2 C . 1 3 t'CSTS £ E Cl C.14C3"ir£2E 0 1 ~CT<TE 5 7TE~8~F. (T3 -6.221 S T T T O T ^ C.260',-;<;S8F-02 0.127fc9V95E 01 0.131E470CE 01 C.5C612C27E C3 -0.414 7C52 8E-01 0.23299S96E-C2 0.122E9991E Cl 0. 12293C24E C l C . 52 75 9 741 E 03 -0.30326843E-03 0.2C629999E-02 0. 1 1529993C C l 0.114C2241E C l C.55275513E C3 0. 13665199E-01 0. I 7549999E-02 0.1C589991E 01 0.10337248E 01 C . 5 8 9 0 K 8 5 E C3 0 . 25 2 74 2 7 7E-C1 0. 1645J000E-02_0^99 1999S8E CO C.9943CC78F PC 0.6044379SE 03 -0.23007989F-02 0. 1597"9998E r02 1 . 911 99999 E 00 C.97719324E 00 C.61T51CC1F C2 C. 48 C6 7 5~7CE-C2 0.131C0CCCE-O2 C.EE399994F CO 0.86311399E 00 C.66268237E 03 0 . 15 e £ 594 9E-01 0.125200C0E-02 C.E5C99995F CC C.345C7C78E 00 C.67497656E C3 0.5929172OE-02 0. 1220 r;oOOE-02 O.E4(99994F 00 0 .332 17019E 00 C.682 10669c C3 C. 14 £ 2 9 75 5E-Cl 0. 1223999S6-C2 0.E3699995E 00 0.83379012E 00 0.68120117E 03 0.32098293E-02 0. 1254 9 9 98E-02 C f 32 9_99 94E CC C.846273C6E PC C . £ 74321 C 4E C3 -0. 13273120E-01 C. e?9999"t)UE-03 0 . £~S 799999 E CO C.6605 1 173E 00 C.79579726F C3 - 0. 25 1 1 739 7E-02 C.£7£99<:e2E-03 0.5£4999 c.£t CC C.5E3E286EE CC C.85939453F C3 0. 147 11022E-02 0.615'<9980E-03 0.55299997E PC C.54E7667SF CC C.69CB5547E C2 0. 42331 t l 5E-C2 C.46C9997SE-03 C . 459S9«93E 00 0.45416898F 00 C.98518262E 03 0.582 1CC32E-02 0.45299996E-C2 0.42C99994E CO C.448E5677E OC C.99085376E 03 -0.27856827E-01 ~~STCP O'- ' EXECLTICN TERMINATED ISICNCFF 217 3. I n t e r a c t i o n of P r e s e r v a t i v e Mixtures w i t h Cetomacrogol (a) C a l c u l a t i o n of a t h e o r e t i c a l b i n d i n g curve using Eq.30 ( i ) Determine the i n t e r a c t i o n of a p r e s e r v a t i v e (D) w i t h ceto-macrogol. C a l c u l a t e b i n d i n g parameters n^, n^, and using the method described i n appendix 2. ( i i ) Determine the i n t e r a c t i o n of another p r e s e r v a t i v e (C), the competitor, w i t h cetomacrogol. C a l c u l a t e b i n d i n g parameters n^, n^, K ^ and using the method described i n appendix 2. ( i i i ( i i i ) Determine the bindin g of a v a r i a b l e c o n c e n t r a t i o n of D w i t h cetomacrogol i n presence of a constant c o n c e n t r a t i o n of C. C a l c u l a t e values of [ D f ] , [ D b ] , [ C f ] and [ x ^ ] . ( i v ) S u b s t i t u t e values of n^, n^, Kjl» K d 2 ' K c l ' K c 2 ' ^ D f ^ a n c* [C^] i n t o Eq.30 and c a l c u l a t e r . (y) C a l c u l a t e the value of r / [ D ^ ] . ( v i ) P l o t r/[D^] versus r (Scatchard p l o t ) . (b) Sample c a l c u l a t i o n s f o r the i n t e r a c t i o n of p r e s e r v a t i v e mixtures w i t h cetomacrogol using the data f o r .the i n t e r a c t i o n of c h l o r o c r e s o l w i t h cetomacrogol i n absence and presence of a constant c o n c e n t r a t i o n of methyl paraben ( F i g . 20, curves A and B). 218 (i) Interaction of chlorocresol with cetomacrogol (Fig. 20, curve A) moles 1 ^ x 103 - r/[D f] [M] 14.86 4.58 1.93 421.49 11.03 3.24 1.43 443.07 10.44 2.88 1.36 471.03 8.27 2.01 1.07 534.66 8.33 2.00 1.08 540.45 5.92 1.18 0.77 652.43 5.91 1.15 0.76 666.48 3.19 0.45 0.41 920.44 M = 7.69 x 10 moles 1 n± = 0.5334 K d l = 2549.5 n = 243.27 K =1.2789 ( i i ) Interaction of methyl paraben with cetomacrogol (Fig. 23, curve A). moles 1 ^ r/[C f] Cc f] M : i 8.26 11.63 1.07 92.33 6.89 9.10 0.89 98.54 5.77 6.72 0.75 111.64 4.34 4.95 0.56 114,-14 3.19 3.19 0.41 130.04 1.80 1.54 0.234 151.91 M = 7.69 x 10 moles 1 n! = 0.7276 K 1 = 166.62 1 c l ni = 69.47 K „ = 0.7412 2 c2 219 ( i i i ) Interaction of chl o r o c r e s o l with cetomacrogol i n presence of a constant concentration of methyl paraben (Fig. 20, curve B) Experimental values Values c using alculated Eq.30 moles 1 r./[Df] r r/[D f] [ V [D f] 1 " [M] 10.38 3.11 1.35 434.30 1.39 449.07 9.39 2.64 1.22 461.95 1.24 468.56 8.10 2.18 1.05 483.35 1.07 493.97 6.86 1.69 0.89 525.54 0.90 532.13 4.69 1.04 0.61 587.25 0.64 617.64 3.10 0.63 0.40 641.80 0.45 722.81 M = 7.69 x 10 moles 1 Tota l methyl paraben concentration = 8.54 x 10 moles 1 N.B. A l l c a l c u l a t i o n s were performed using IBM 370 computer. (c) Analysis of antagonism between the two d i f f e r e n t types of small molecules f o r binding with a macromolecule using Scatchard p l o t Interaction between a small molecule and-macromolecule can be expressed by Eq.64 which i s a rearrangement of Eq.24 and i s known as the 1 Scatchard equation (Scatchard, 1949). A p l o t r = nK - rK (Eq.64) of Eq.64 gives a st r a i g h t l i n e with a negative slope (Fig. 53,a). Extra-p o l a t i o n of the l i n e to r axis gives the value of the maximum number of free binding s i t e s , n, a v a i l a b l e per mole of macromolecule for the i n t e r -a c t i o n . The slope of the l i n e gives the i n t r i n s i c a s s o c i a t i o n constant, K. The i n t r i n s i c a s s o c i a t i o n constant i s defined as the equilibrium constant 220 r r F i g . 53. Analysis of antagonism using Scatchard p l o t . (a) Interaction i n absence of competitor. (b) Competitive antagonism. (c) Non-competitive antagonism. (d) Mixed competitive-noncompetitive antagonism. A - binding of D with M i n absence of C. B and C -bindingcof D with M i n presence of a constant C. The value of C for curve B i s less than curve C. 221 f o r the a s s o c i a t i o n r e a c t i o n between 1 mole of. f r e e drug and 1 mole of unoccupied b i n d i n g s i t e to form 1 mole of complex. Curvature i n t h i s p l o t i s an i n d i c a t i o n of the e l e c t r o s t a t i c i n t e r a c t i o n s between the bindi n g s i t e s of the macromolecule, the negative or p o s i t i v e cooperative e f f e c t s i n the b i n d i n g , or the involvement of more than one c l a s s of bindi n g s i t e s i n the i n t e r a c t i o n . However, i n the present work, curvature i n the Scatchard p l o t was assumed to be due to the presence of two c l a s s e s of bi n d i n g s i t e s and hence the b i n d i n g data was c h a r a c t e r i z e d using Eg.25. For the sake of s i m p l i c i t y i n the present d i s c u s s i o n only one c l a s s of bi n d i n g s i t e s i s assumed. Antagonism between a small molecule, D, and another small molecule, the competitor, C, f o r the b i n d i n g w i t h a macromolecule, M, can be c l a s s i f i e d i n t o three types. Case I . Competitive antagonism: The molecule C i s s a i d to be competitive w i t h the molecule D i f i t combines r e v e r s i b l y w i t h the same bin d i n g s i t e s of the macromolecule as the molecule*D. However, i f the combination of C w i t h M induced a conformational change that a l t e r e d the bi n d i n g energy of the complex MD, the antagonism would be c l a s s i f i e d as c o m p e t i t i v e ^ even i f C and D combined w i t h M independently a t d i f f e r e n t s i t e s . The e f f e c t of the competition,, i n any case, i s a r e d u c t i o n of the apparent a f f i n i t y of D .for M i n the presence of C, as p r e d i c t e d by the law of mass a c t i o n (Eq.30). In Scatchard p l o t , competitive i n h i b i t i o n i s e x h i b i t e d by r e d u c t i o n of the slope and hence the a s s o c i a t i o n constant, K^, w i t h i n c r e a s i n g concentrations of the competitor, C ( F i g . 53,b). However, n remains constant f o r a l l values of C. 222 Case I I . Noncompetitive antagonism: The molecule C i s said to be noncompetitive i f i t in a c t i v a t e s the binding s i t e s f o r D so that the e f f e c t i v e complex with the molecule D cannot be formed, regardless of the concentration of D. The molecule C might combine i r r e v e r s i b l y with M at the same s i t e where D o r d i n a r i l y combines, giving r i s e to apparent noncompetitive i n t e r a c t i o n . The e s s e n t i a l point i n noncompetitive antagonism i s that the molecule D has no influence upon the degree of antagonism. , In the Scatchard p l o t , noncompetitive antagonism i s exhibited by the reduction i n the value of n.with increasing concentrations of the competitor, C (Fig. 53,c). However, the slope, and hence the a s s o c i a t i o n constant, K, remains constant for a l l values.; of C. Case I I I . Mixed competitive-noncompetitive antagonism: This antagonism i s of a complex nature and their, mechanisms are not well defined. In the Scatchard p l o t , t h i s antagonism i s exhibited by both changes i n the values of n and K with increase i n the concentration of C (Fig. 53,d). 223 4. Sample Calculations for the Interaction of Chlorocresol with Mixtures of some Nonoionic Surfactants (Fig. 30, lowest curve) (a) Binding parameters for the interaction of chlorocresol with Texofor A,,: 16 nJ = 0.5377; Kj = 2122.4 n^ = 141.53; = 1.647 (b) Binding parameters for the interaction of chlorocresol with Texofor k,n: 60 n = 0.4319; K± = 4974.3 n 2 =148.4; K £ = 3.161 (c) Interaction of chlorocresol with mixtures of Texofor A,, and k,n -1 3 moles 1 x 10 Calculated using Eq.37 and values of Experimental n ^ n 2, , K£, [Mj], [M I X] and [D f] : D>f] [D t] [Bfc] \ 3.694 17.19 18.21 2.667 13.34 13.99 2.039 10.87 11.37 1.422 8.347 8.719 0.847 6.047 6.099 0.358 3.308 3.473 Concentration of Texofor A,,, [MT] = 2.114 x 10 moles 1 16 I -3 Concentration of Texofor A^n, '[iMTT] = 5.51 x 10 moles 1 N.B. A l l calculations were performed using IBM 370 computer. 224 5 . Calculation of Percent Loss of Preservative From the Aqueous Phase of Surfactant Solution for a Given Loss of Preservative From Total System 1. Let [Dj.] and _D-] be the i n i t i a l total and free preservative concentrations respectively. 2. Let loss in the concentration of preservative from the surfactant solution = [ l - t ^ ] . 3. Concentration of Preservative remaining in the surfactant solution - [ » J - [ D t J . C D ' ] . 4 . Concentration of free preservative in the aqueous phase, [D^], for a given total preservative concentration, _D^], was calculated using Eq .64 which is. a rearrangement of Eq.28. K - K g L " . ' ] 3 + (K- + K- + n-K-.K-M + n . K - K - M - K.K^D^]) [D^] 2 + (1 + n.K - M + n 2K 2[M] - K-DBL'J.-] - K-LDj]) [ D £ ] - [ D ' ] = 0 (Eq .65) The roots of Eq .64 were calculated using Bairstow's method (U.B.C. computer program, ZPOLY). 5 . Percent preservative loss from the aqueous phase of surfactant = [100([D f] - [Dp)j] / [ D F ] . 225 6. C a l c u l a t i o n of Percent Loss of P r e s e r v a t i v e From the Aqueous Phase of an Emulsion f o r a Given Loss of P r e s e r v a t i v e From T o t a l System A s i m i l a r method i s used as given i n Appendix 5 f o r a s u r f a c t a n t s o l u t i o n , except [D^] f o r a given [D^] i s c a l c u l a t e d using Eq.65 (Bean, Heman-Ackah and Thomas, 1965). [ D ' ] ( q + 1 ) [DI] = — J (Eq.66) K q + 1 w 226 7. Calculation of Percent Loss of Preservative From the Aqueous Phase of an Emulsion Stabilized with a Surfactant - For a Given Loss of Preservative From Total System.. A similar method is used as given in Appendix 3 for a surfactant solution, except [D^] for a given _D£ ] is calculated using Eq.66 which is a rearrangement of Eq.48. The roots of Eq.65 were calculated using Bairstow's method (U.B.C. computer program, ZPOLY). ( K - K . + K°q K . K - ) [ D ^ ] 3 + (K- + K 2 + n.K.K^M] + n^-K^M] + K°q K- + K°q K 2 - K.K 2 [D [ ] q - K.K^D ' ] ) [ D ^ ] 2 + (1 + n-K-M + n ^ M + K°q - K - [ D y q - K j C D ' ] - K 2 [D^] q - K 2 [ D » ] ) [ D £ ] - [D^] (q + 1) = 0 (Eq.67) 

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