21 P PROBLEMS I N THE DETERMINAT ON O F PHENOLIC PKA VALUES BY SPECTROPHOTOMETRY K . C . James and A. R i c h a r d s , We s h School o f Pharmacy, UWIST, Cathays P a r k , C a r d i f f , CF1 3NU.
P r e c i s e d i s s o c i a t i o n c o n s t a n t s of some p h e n o l i c b i b e n z y l s were r e q u i r e d a s p a r t of a QSAR i n v e s t i g a t i o n . A U. v. s p e c t r o p h o t o m e t r i c t e c h n i q u e was employed, depending on d i s s o c i a t e d and u n d i s s o c i a t e d s p e c i e s absorbi.ng a t d i f f e r e n t wavelengths. The p r o c e d u r e i s d e s c r i b e d i n d e t a i l by A l b e r t R S e r j e a n t ( 1 9 7 1 ) . I n p r e l i m i n a r y e x p e r i m e n t s w i t h h e x o e s t r o l and s t i l b o e s t r o l , 3 b u f f e r combinatiors were used, borax/KOH, Na c0 / N a H C 0 3 and butylamine/HCl, p r o v i d i n g 7 b u f f e r 2 3 The pKa v a l u e s were s o l u t i o n s , c o v e r i n g a range of pH v a l u e s from 9.4 t o 1 1 . 4 . r e p r o d u c i b l e a t each i n d i v i d u a l pH, b u t appeared t o i n c r e a s e p r o g r e s s i v e l y Determinations w i t h i n c r e a s i n g pH. The change was s l b s e q u e n t l y l i n k e d w j t h t i m e . had always been c a r r i e d O u t s t a r t i n g w i t h t h e l o w e s t pH m i x t u r e and working up When t h e sequence was a l t e r e d , a d i f f e r e n t pKa/pH p r o f i l e t h e pH s c a l e . emerged, t h e pK v a l u e s changing w i t h t h e time i n t e r v a l between p r e p a r a t i o n o f A u t o x i d a t i o n , which i s known t o o c c u r s o l u t i o n and me2surement of absorbance. i n aqueous s o l u t i o n s of p h e n o l s , was t h e r e f o r e s u s p e c t e d . The r e a c t i o n i s accompanied by formation of quinones (Kenyhercz & K i s s i n g e r , 1 9 7 8 ) , which a r e s t r o n g e r chromophores t h a n p h e n o l s . The absorbances of t h e s y n t h e t i c oestrogen s o l u t i o n s i n c r e a s e d w i t h t i m e , commensurate w i t h t h i s , b u t t h e r a t e o f i n c r e a s e d e c r e a s e d w i t h i n c r e a s i n g pH, u n t i l a t pH v a l u e s i n t h e r e g i o n of 1 3 ( 0 . 1 M NaOH) t h e absorbance remained c o n s t a n t . The observed pK v a l u e s m u s t t h e r e f o r e have been dependent on t h e pH o f t h e medium, a s Well 2s t h e time i n t e r v a l between p r e p a r a t i o n o f s o l u t i o n and measurement of absorbance. V a r i a t i o n i n t h e r a t e of a u t o x i d a t i o n of phenols w i t h pH h a s been n o t e d b e f o r e (Beynon & James, 1 9 6 7 ) and a t t r i b u t e d t o t h e u n i o n i s e d phenol b e i n g t h e r e a c t i v e s p e c i e s . The above o b s e r v a t i o n s f i t t h i s s u g g e s t i o n . The absorbances reached l i m i t i n g v a l u e s , assumed t o r e p r e s e n t e x h a u s t i o n of t h e d i s s o l v e d oxygen. When t h e measurements were r e p e a t e d u s i n g f r e s h l y b o i l e d and cooled w a t e r , and i n an atmosphere o f n i t r o g e n , more s t a b l e r e a d i n g s were o b t a i n e d , b u t t h e d e c l i n e i n a b s o r p t i o n w i t h t i m e was n o t completely e l i m i n a t e d . More d r a s t i c methods o f removing oxygen a r e t h e r e f o r e n e c e q s a r y . A n a l t e r n a t i v e approach was a t t e m p t e d , whereby p l o t s of absorbance a g a i n s t time were e x t r a p o l a t e d back t o z e r o t i m e . Curve f i t t i n g c a l c u l a t i o n s i n d i c a t e d t h a t t h e two v a r i a b l e s were b i n o m i a l l y r e l a t e d , s o t h a t absorbances a t z e r o time c o u l d be o b t a i n e d from t h e c h a r a c t e r i s t i c q u a d r a t i c e a u a t i o n s . The r e s u l t s There was no d r i f t w i t h pH, y i e l d e d pK v a l u e s which were independent o f t i m e . and t h e r e p a r o d u c i b i l i t y was s i g n i f i c a n t l y improved.
The e x p e r i e n c e s e n c o u n t e r e d h e r e s u g g e s t t h a t some p r e v i o u s l y r e p o r t e d p h e n o l i c pKa v a l u e s may n o t be a s p r e c i s e a s i s b e l i e v e d . A l b e r t . A . 81 S e r j e a n t . E . P . ( 1 9 7 1 ) I n The Determination o f I o n i z a t i o n C o n s t a n t s , pp 44-64. London: Chapman and H a l l . (1967) J.Pharm.Pharmaco1. 1 9 , 660-666 Beynon. C . B . R James. K . C . 1-2 Kenyhercz. T.M. 81 K i s s i n g e r . P . T . (1978). J.Anal.Toxico1.
2,
P r e c i s e d i s s o c i a t i o n c o n s t a n t s of some p h e n o l i c b i b e n z y l s were r e q u i r e d a s p a r t of a QSAR i n v e s t i g a t i o n . A U. v. s p e c t r o p h o t o m e t r i c t e c h n i q u e was employed, depending on d i s s o c i a t e d and u n d i s s o c i a t e d s p e c i e s absorbi.ng a t d i f f e r e n t wavelengths. The p r o c e d u r e i s d e s c r i b e d i n d e t a i l by A l b e r t R S e r j e a n t ( 1 9 7 1 ) . I n p r e l i m i n a r y e x p e r i m e n t s w i t h h e x o e s t r o l and s t i l b o e s t r o l , 3 b u f f e r combinatiors were used, borax/KOH, Na c0 / N a H C 0 3 and butylamine/HCl, p r o v i d i n g 7 b u f f e r 2 3 The pKa v a l u e s were s o l u t i o n s , c o v e r i n g a range of pH v a l u e s from 9.4 t o 1 1 . 4 . r e p r o d u c i b l e a t each i n d i v i d u a l pH, b u t appeared t o i n c r e a s e p r o g r e s s i v e l y Determinations w i t h i n c r e a s i n g pH. The change was s l b s e q u e n t l y l i n k e d w j t h t i m e . had always been c a r r i e d O u t s t a r t i n g w i t h t h e l o w e s t pH m i x t u r e and working up When t h e sequence was a l t e r e d , a d i f f e r e n t pKa/pH p r o f i l e t h e pH s c a l e . emerged, t h e pK v a l u e s changing w i t h t h e time i n t e r v a l between p r e p a r a t i o n o f A u t o x i d a t i o n , which i s known t o o c c u r s o l u t i o n and me2surement of absorbance. i n aqueous s o l u t i o n s of p h e n o l s , was t h e r e f o r e s u s p e c t e d . The r e a c t i o n i s accompanied by formation of quinones (Kenyhercz & K i s s i n g e r , 1 9 7 8 ) , which a r e s t r o n g e r chromophores t h a n p h e n o l s . The absorbances of t h e s y n t h e t i c oestrogen s o l u t i o n s i n c r e a s e d w i t h t i m e , commensurate w i t h t h i s , b u t t h e r a t e o f i n c r e a s e d e c r e a s e d w i t h i n c r e a s i n g pH, u n t i l a t pH v a l u e s i n t h e r e g i o n of 1 3 ( 0 . 1 M NaOH) t h e absorbance remained c o n s t a n t . The observed pK v a l u e s m u s t t h e r e f o r e have been dependent on t h e pH o f t h e medium, a s Well 2s t h e time i n t e r v a l between p r e p a r a t i o n o f s o l u t i o n and measurement of absorbance. V a r i a t i o n i n t h e r a t e of a u t o x i d a t i o n of phenols w i t h pH h a s been n o t e d b e f o r e (Beynon & James, 1 9 6 7 ) and a t t r i b u t e d t o t h e u n i o n i s e d phenol b e i n g t h e r e a c t i v e s p e c i e s . The above o b s e r v a t i o n s f i t t h i s s u g g e s t i o n . The absorbances reached l i m i t i n g v a l u e s , assumed t o r e p r e s e n t e x h a u s t i o n of t h e d i s s o l v e d oxygen. When t h e measurements were r e p e a t e d u s i n g f r e s h l y b o i l e d and cooled w a t e r , and i n an atmosphere o f n i t r o g e n , more s t a b l e r e a d i n g s were o b t a i n e d , b u t t h e d e c l i n e i n a b s o r p t i o n w i t h t i m e was n o t completely e l i m i n a t e d . More d r a s t i c methods o f removing oxygen a r e t h e r e f o r e n e c e q s a r y . A n a l t e r n a t i v e approach was a t t e m p t e d , whereby p l o t s of absorbance a g a i n s t time were e x t r a p o l a t e d back t o z e r o t i m e . Curve f i t t i n g c a l c u l a t i o n s i n d i c a t e d t h a t t h e two v a r i a b l e s were b i n o m i a l l y r e l a t e d , s o t h a t absorbances a t z e r o time c o u l d be o b t a i n e d from t h e c h a r a c t e r i s t i c q u a d r a t i c e a u a t i o n s . The r e s u l t s There was no d r i f t w i t h pH, y i e l d e d pK v a l u e s which were independent o f t i m e . and t h e r e p a r o d u c i b i l i t y was s i g n i f i c a n t l y improved.
The e x p e r i e n c e s e n c o u n t e r e d h e r e s u g g e s t t h a t some p r e v i o u s l y r e p o r t e d p h e n o l i c pKa v a l u e s may n o t be a s p r e c i s e a s i s b e l i e v e d . A l b e r t . A . 81 S e r j e a n t . E . P . ( 1 9 7 1 ) I n The Determination o f I o n i z a t i o n C o n s t a n t s , pp 44-64. London: Chapman and H a l l . (1967) J.Pharm.Pharmaco1. 1 9 , 660-666 Beynon. C . B . R James. K . C . 1-2 Kenyhercz. T.M. 81 K i s s i n g e r . P . T . (1978). J.Anal.Toxico1.
2,
