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Hydrogenation of ∝- oximinoglutaric acid on Raney Nickel : studies toward optical resolution McGinnis, Michael J. 1977

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HYDROGENATION OF <*-OXIMINOGLUTARIC ACID ON RANEY NICKEL: STUDIES TOWARD OPTICAL RESOLUTION BY MICHAEL J . McGINNIS B.Sc., 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 , 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D e p a r t m e n t o f C h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA, O c t o b e r , 1977 © M i c h a e l M c G i n n i s In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f i nanc ia l gain sha l l not be allowed without my writ ten pe rm i ss i on . Department of C h e m i s t r y The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date O c t o b e r 13, 1977 - i i -ABSTRACT Th i s t h e s i s d e s c r i b e s p h y s i c o c h e m i c a l s t u d i e s to i n v e s t i g a t e a r e p o r t of spontaneous o p t i c a l r e s o l u t i o n i n the h y d r o g e n a t i o n of k e t o g 1 u t a r i c a c i d and i t s oxime by some Japanese worker s i n 1958. S i n c e the i n t e n t i o n was to s tudy the k i n e t i c s of the r e a c t i o n , a r a p i d method f o r q u a n t i t a t i v e a n a l y s i s of r e a c t a n t and p r oduc t i n aqueous s o l u t i o n was r e q u i r e d , and a l a r g e p a r t of the t h e s i s i s concerned w i t h the deve lopment of s u i t a b l e a n a l y t i c a l methods. For the r e d u c t i o n of the k e t o a c i d , s e v e r a l methods were s t u d i e d and a l l found u n s u i t a b l e . For the p r o d u c t i o n of g l u t a m i c a c i d from the ox ime, a method u s i n g the r e a c t i o n of the am inoac i d w i t h n i n h y d r i n was q u i t e s u c c e s s f u l . The h y d r o g e n a t i o n s were c a t a l y s e d by Raney N i c k e l , and the work r e q u i r e d s tudy of the s u r f a c e a rea of the c a t a l y s t and of i t s c h a r a c t e r i s t i c s f o r c o m p e t i t i v e a d s o r p t i o n of r e a c t a n t , p r oduc t and phosphate b u f f e r from the r e a c t i o n m i x t u r e . The k i n e t i c s t u d i e s showed r a t h e r low r e p r o d u c i b i l i t y . In a number of c a s e s , the r e a c t i o n proceeded i n a marked l y a u t o c a t a l y t i c manner, g i v i n g p r oduc t i n p r o p o r t i o n to the square of the t i m e . In o t h e r c a s e s , the r e a c t i o n cu rve was c l o s e to l i n e a r ( z e r o - o r d e r ) . Except i n one case which gave an o p t i c a l e f f e c t of m a r g i n a l s i g n i f i c a n c e , no spontaneous r e s o l u t i o n was o b s e r v e d , n e i t h e r c o u l d o p t i c a l r e s o l u t i o n be induced by dop ing the system w i t h an enan t i omer o f the p r o d u c t . - i i i -I c onc l ude t h a t a u t o c a t a l y t i c b e h a v i o r wh ich might p r o v i d e the nece s s a r y c o n d i t i o n s f o r spontaneous r e s o l u t i o n o c cu r s i n the h y d r o g e n a t i o n , but t h a t my r e s u l t s do not s u b s t a n t i a t e the Japanese w o r k e r s ' c l a i m t h a t spontaneous r e s o l u t i o n o c c u r s . - i v -TABLE OF CONTENTS Page T I T L E PAGE ABSTRACT TABLE OF CONTENTS LI S T OF TABLES L I S T OF FIGURES ACKNOWLEDGEMENTS I . INTRODUCTION — THEORY AND OUTLINE OF EXPERIMENTAL WORK T h e o r y O u t l i n e o f E x p e r i m e n t a l Work (1) o { - K e t o g l u t a r i c A c i d and ©<-Hydroxyglutaric Ac i d (2) G l u t a m i c A c i d and o d - O x i m i n o g l u t a r i c A c i d ~ 10 (3) Raney N i c k e l 11 (4) A d s o r p t i o n S t u d i e s 11 (5) H y d r o g e n a t i o n Runs '12 I I . MATERIALS AND ANALYTICAL METHODS 16 (1) - K e t o g l u t a r i c A c i d and < ^ - H y d r o x y g l u t a r i c A c i d 16 T a c t i c s f o r D e t e r m i n a t i o n o f S m a l l Amounts o f H y d r o x y a c i d i n L a r g e Q u a n t i t i e s o f K e t o a c i d 19 A l d e h y d e D e t e r m i n a t i o n v i a S c h i f f R e a g e n t 20 D e t e r m i n a t i o n o f H y d r o x y a c i d by N.M.R. 24 D e t e r m i n a t i o n o f H y d r o x y a c i d by B l e a c h i n g o f Reduced M o l y b d a t e 36 P e r m a n g a n a t e T i t r a t i o n 42 i i i i v v i v i i - V Page (2) G l u t a m i c A c i d - c ^ - - O x i m i n o g l u t a r i c A c i d 53 D e t e r m i n a t i o n o f G l u t a m i c A c i d by N i n h y d r i n Method 56 (3) Raney N i c k e l : C h a r a c t e r i s t i c s and H a n d l i n g 64 I I I . ADSORPTION STUDIES ON RANEY NICKEL 72 IV. HYDROGENATION RUNS 87 O p e r a t i o n of H y d r o g e n a t o r and P o l a r i m e t e r 87 H y d r o g e n a t i o n P r o c e d u r e 92 H a n d l i n g of Samples to A v o i d Mold Growth 99 E v i d e n c e o f S i d e R e a c t i o n 99 E x p e r i m e n t a l R e s u l t s 102 BIBLIOGRAPHY 119 APPENDIX 120 - v i -LIST OF TABLES T a b l e Page 1. Summary of Hydrogenation Conditions and Results 105 - v i i -LIST OF FIGURES Figure Page-; I. . °£ -Hydroxyglutaric Acid Na Salt: Optical Rotations vs. Hyroxyacid Concentration 18 2. Calibration of Schiff Reagent: Batch One 23 3. Calibration of Schiff Reagent: .Batches One and Two 25 4. N.M.R. Spectrum of «V-Ketoglutaric Acid 27 5. N.M.R. Spectrum of oOHydroxyglutaric Acid Na Salt 28 6. N.M.R. Spectrum of Succinic Acid 29 7. N.M.R. Spectrum of Pb(OAc)^ + •V-Hydroxyglutaric Acid 30 8. N.M.R. Spectrum of Succinic Acid and Succinaldehydic Acid 31 9. N.M.R. Calibration Curve: Relative Peak Area vs. I n i t i a l [Hydroxyacid] 33 10. N.M.R. Spectrum of Pb(OAc)^ + »£-Ketoglutaric Acid 35 I I . Molybdate Blue Calibration: Hydroxyacid 39 12. Molybdate Blue Calibration: Ketoacid 41 13. Permanganate T i t r a t i o n Calibration 44 14. N.M.R. Spectrum of Unheated °^-Ketoglutaric Acid 46 15. N.M.R. Spectrum of Unheated o£-Ketoglutaric Acid Na Salt 47 16. N.M.R. Spectrum of Reacidified Product 48 17. Potentiometric T i t r a t i o n : Ketoacid 50 18. Potentiometric T i t r a t i o n : Reacidified Ketoacid 51 19. Glutamic Acid Optical Rotation vs. pH (At High pH) 55 20. Glutamic Acid Optical Rotation vs. pH (Low pH) 57 21. Ninhydrin Calibration Curve for Glu/Oxime: 0 - 100 % 61 22. Ninhydrin Calibration Curve for Glu/Oxime: 0 - 10 % 62 23. Raney Nickel N 0 Adsorption Isotherm 67 - v i i i -Figure Page 24. BET Plot f o r N 2 Adsorption on Raney Nickel 68 25. Isotherms of Glu, Phos and Ox Adsorption on Raney N i c k e l 75 26. Langmuir Plot f o r Glu Adsorption on Raney Ni c k e l 77 27. BET Plot for Glu Adsorption on Raney Ni c k e l 78 28. E f f e c t of [Phos] on the Ninhydrin - Glu Reaction 82 29. Adsorption of 0.001 M Glu on Raney Ni c k e l i n Presence of Varying [Phos] 84 30. High Pressure Hydrogenator: Schematic Diagram 88 31. High Pressure Hydrogenator: Reaction Vessel 90 32. Fischer Proportional Temperature Control C a l i b r a t i o n Curves 95 33. O p t i c a l Y i e l d vs. Chemical Y i e l d at r = 0.002° 98 34. O.R.D. Spectra of Glutamic Acid and °£ -Hydroxyglutaric Acid 103 35. Run 3(a): Chemical Y i e l d vs. Time 109 36. Run 3(a): O p t i c a l Y i e l d vs. Time 110 37. Run 3(b): Chemical Y i e l d vs. Time i l l 38. Run 9Xa): Chemical Y i e l d vs. Time 113 39. Run 9(b): Chemical Y i e l d vs. Time 115 40. Run 9(b): Op t i c a l Y i e l d vs.: Time 116 - i x -ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k D r . Hayward f o r d e t e r m i n i n g t h e O.R.D. s p e c t r a . A l s o , t h e h e l p o f D r . R o s e n t h a l and t h e members o f h i s r e s e a r c h g r o u p i n f i n d i n g p a r t s and i n i n s t r u c t i n g me i n t h e u s e of t h e h y d r o g e n a t o r was much a p p r e c i a t e d . B r i n P o w e l l was most h e l p f u l on s e v e r a l o c c a s i o n s when m e c h a n i c a l d i f f i c u l t i e s a r o s e . Dr. J i m S c o t t ' s a s s i s t a n c e i n t h e s u r f a c e a r e a d e t e r m i n a t i o n o f Raney N i c k e l was a p p r e c i a t e d as was h i s o v e r a l l h e l p i n t h e l a b . D r . Yosh'i K o g a , who e f f e c t e d t h e t r a n s l a t i o n o f t h e J a p a n e s e work n e c e s s a r y and p r e r e q u i s i t e t o my work, was t h e s o u r c e o f v a l u e d a d v i c e and i n t e r e s t i n t h e c o u r s e o f t h i s w o r k . F i n a l l y , t h e p e r s o n most d i r e c t l y c o n n e c t e d t o my work -- my R e s e a r c h S u p e r v i s o r , D r . L. G. H a r r i s o n . H i s i n s i g h t , p a t i e n c e and humour were o u t s t a n d i n g f a c t o r s i n c o n t r i b u t i n g t o my a p p r e c i a t i o n o f t h e work o f t h e l a s t f o u r y e a r s . - 1 -I . INTRODUCTION THEORY AND OUTLINE OF EXPERIMENTAL WORK Amino a c i d s o f one c h i r a l i t y o n l y a r e f o u n d i n l i v i n g s y s t e m s , n o t t h e r a c e m i c m i x t u r e t h e r m o d y n a m i c a l l y s t a b l e and e x p e c t e d . I was drawn t o t h i s a r e a o f work by an i n t e r e s t i n why t h i s m i g h t be s o . The f i r s t q u e s t i o n one f a c e s i s w h e t h e r : (1) t h e r e i s a p r e f e r e n c e f o r e n a n t i o -m e r i s m o f one h a n d e d n e s s i . e . t h a t one i s more s t a b l e t h a n t h e o t h e r o r (2) asymmetry a r o s e as an a c c i d e n t i . e . t h e r e i s no o v e r a l l a s ymmetry i n t h e u n i v e r s e w h i c h a c t s s i g n i f i c a n t l y a t t h e b i o l o g i c a l s c a l e . (1) The f i r s t v i e w p o i n t i s p h y s i c a l : t h a t a s y m m e t r i c p h y s i c a l p r o c e s s e s e x i s t e.g. c h i r a l a s ymmetry i n ^ - d e c a y and l o c a l i m b a l a n c e i n i n t e n s i t y o f o p p o s i t e l y handed c i r c u l a r l y p o l a r i s e d r a d i a t i o n , w h i c h a r e s u f f i c i e n t i n t h e s h o r t r u n p o s s i b l y t o e f f e c t r e s o l u t i o n o f a s i m p l e a c h i r a l —^ c h i r a l r e a c t i o n . A l t e r n a t i v e l y o v e r a p e r i o d o f t i m e a s m a l l p r e f e r e n c e f o r e n a n t i o m e r s o f one c h i r a l i t y w o u l d be a m p l i f i e d by t h e l a r g e number of c h e m i c a l r e a c t i o n s p r e r e q u i s i t e t o a . l i v i n g s y s t e m , r e s u l t i n g i n a g r a d u a l p r e f e r e n c e f o r c h i r a l l i f e . T h i s a p p r o a c h has been a d v a n c e d ' -mos11 y be p h y s i c i s t s and work has been done (1) i n d i c a t i n g some p r e f e r e n c e f o r one e n a h t i o m e r i n a r e d u c -t i o n r e a c t i o n f r o m a p r o c h i r a l c h i r a l s p e c i e s u n d e r t h e c o n d i t i o n o f i n t e n s e r a d i o a c t i v i t y ( - d e c a y ) . - 2 -(2) The second t r e a t m e n t i s c h e m i c a l . I t seeks to f i n d k i n e t i c cond i t i ons .meces s a r y ' f or ;t he:.- s tab i 1 i t y of«a r e s o l v e d s teady s t a t e . The o n l y i n f l u e n c e s needed to t r i g g e r the change r a cem ic -> r e s o l v e d would be ones t h a t a re n o r m a l l y n o n - c h i r a l , e . g . g r a d i e n t s of p r o p e r t i e s such as t empe ra tu re o r random f l u c t u a t i o n s on a m i c r o -s c o p i c l e v e l . These f a c t o r s c o u l d be c h i r a l o n l y , i n a s en se , by " a c c i d e n t " . Thermodynamica11y t h e r e s hou l d be-no p r e f e r e n c e f o r one enan t i omer ; ..the v racemi c e s t a t e i s approached a t e q u i l i b r i u m i n l a b o r a t o r y r e a c t i o n s . T h i s approach a s s e r t s t h a t r e s o l u t i o n , when i t o c c u r s , s h o u l d show no e n e r g e t i c p r e f e r e n c e f o r one e n a n t i o m e r i c outcome over the o t h e r . The f o r emos t r e q u i r e m e n t i s d e v e l o p i n g n e c e s s a r y k i n e t i c c o n d i t i o n s and t e s t i n g r e a c t i o n systems t o see i f a r e s o l v e d s t eady s t a t e can be a c h i e v e d and i f the systems t e s t e d r e a c t e d w i t h t he k i n e t i c s proposed as ne ce s s a r y f o r spontaneous r e s o l u t i o n . The p a r t i c u l a r t r e a t m e n t i n t h i s l a b o r a t o r y of the c h e m i c a l p roce s s l e a d i n g to r e s o l u t i o n d e a l t , f i r s t , w i t h i d e n t i f y i n g nece s s a r y k i n e t i c c o n d i t i o n s under w h i c h , g i v e n a s l i g h t i n i t i a l asymmetry i n e n a n t i o m e r i c p r oduc t m o l e c u l e s , a tendency to a m p l i f y t h i s asymmetry i s found ( H a r r i s o n 1974) . Th i s s i d e s t e p s :the o r i g i n of the i n i t i a l asymmetry which can be s u p p l i e d " a c c i d e n t a l l y " th rough a random - 3 -f l u c t u a t i o n or as a r e s u l t of s l i g h t u n i v e r s a l asymmetry e f f e c t s on a r e a c t i o n . A r e s o l v e d system can a r i s e , t h e n , (1) from u n i v e r s a l c h i r a l i n f l u e n c e s or (2) from l o c a l f l u c t u a t i o n s g i v i n g a c h i r a l e f f e c t . For the purposes of t h i s work "spontaneous r e s o l u t i o n " i s d e f i n e d as r e s u l t i n g o n l y from i n e s c a p a b l e g r a d i e n t s and f l u c t u a t i o n s w h i l e " f o r c e d r e s o l u t i o n " r e s u l t s from the f i r s t i n f l u e n c e . The k i n e t i c model and i t s e x p e r i m e n t a l v i n d i c a t i o n can demonstrate t h a t a p o s s i b l e pathway e x i s t s f o r spon-taneous r e s o l u t i o n . Demonstration of spontaneous asymmetry would r e q u i r e t h a t s u f f i c i e n t e xperiments be performed to show s t a t i s t i c a l l y t h a t a symmetric d i s t r i b u t i o n ( G a u s s i a n ) around the racemic r a t i o i s o b t a i n e d . Any e x p e r i m e n t a l d e m o n s t r a t i o n would be u n c e r t a i n because of the i n e s c a p a b l e p o s s i b i l i t y of minute asymmetric c o n t a m i n a n t s . However, g i v e n a s m a l l i n i t i a l d i s t u r b a n c e , whether s u p p l i e d a c c i d e n t a l l y or through the a d d i t i o n of a c h i r a l seed ( e . g . s m a l l amount of e n a n t i o m e r i c a l l y pure g l u t a m i c a c i d ) the k i n e t i c s of the e x p e r i m e n t a l system may be d e t e r m i n e d . The work r e p o r t e d i n t h i s t h e s i s a r i s e s from two i n v e s t i g a t i o n s by o t h e r workers i n v o l v i n g a l a r g e number of experiments i n t e n d e d to demonstrate spontaneous r e s o l u -t i o n and from a k i n e t i c study by H a r r i s o n on the n e c e s s a r y c o n d i t i o n s f o r achievement of spontaneous r e s o l u t i o n . Work by P i n c o c k (2) on the r e c r y s t a l l i s a t i o n of 1-1 b i n a p h t h y l r e s u l t e d , over a l a r g e number (200) of e x p e r i m e n t s , i n a Gaussian d i s t r i b u t i o n symmetric around z e r o asymmetry. - 4 -H 2 H a r r i s o n showed t h a t these r e s u l t s were c o n s i s t e n t w i t h an 8 c o i n - f l i p d i s t r i b u t i o n , c o r r e s p o n d i n g to 8 n u c l e i of c r y s t a l l i s a t i o n . A c l a i m f o r spontaneous asymmetry was made by Isoda e t . a l . (3) where o ( - k e t o g l u t a r i c a c i d and i t s oxime were reduced under a v a r i e t y of s o l v e n t , pH, t e m p e r a t u r e , c o n d i t i o n s to o < - h y d r o x y g l u t a r i c a c i d and g l u t a m i c a c i d r e s p e c t i v e l y . The paper i n which t h i s work appeared was p u b l i s h e d i n Japanese o n l y and the t r a n s l a t i o n k i n d l y s u p p l i e d by Dr. Y. Koga appears as an appendix. Although t h e r e was some e x p e r i m e n t a l s u pport f o r spontaneous r e s o l u t i o n , t h i s c o n c l u s i o n was weakened by l a c k of numerous experiments f o r a p a r t i c u l a r s e t of c o n d i t i o n s and by what may be c h i r a l c ontaminants r e f l e c t e d i n t h e i r o b s e r v a t i o n t h a t use of p o l a r and no n - p o l a r s o l v e n t s r e s u l t e d i n c h i r a l i t y of o p p o s i t e s i g n s . Theory The f i r s t i n v e s t i g a t i o n l e d to H a r r i s o n ' s i d e a s on a p o s s i b l e method of a c h i e v i n g spontaneous r e s o l u t i o n i n b i o c h e m i c a l e v o l u t i o n w i t h an analogous e x p e r i m e n t a l system i n v o l v i n g heterogeneous c a t a l y s i s of r e d u c t i o n of a p r o c h i r a l s o l u t e adsorbed on a s o l i d c a t a l y s t . The k i n e t i c r e q u i r e m e n t s f o r the achievement of a r e s o l v e d s t a t e were s t a t e d by M i l l s and developed by Frank, S e e l i g , Dekker, Hochstim and H a r r i s o n ( 4 - 8 ) . A review of H a r r i s o n ' s development w i l l o u t l i n e the p o i n t s t h a t the e x p e r i m e n t a l work sought to v e r i f y . - 5 -In H a r r i s o n ' s development the r e a c t i o n scheme i s where L and D r e p r e s e n t c o n c e n t r a t i o n s of r e s p e c t i v e enantiomers and a d s o r p t i o n s a t u r a t i o n i s assumed so t h a t <|>& and are f u n c t i o n s of D/(D + L) and L/(D + L ) . Assuming complete a u t o c a t a l y s i s (no a c t i v i t y w i t h o u t p r oduct a d s o r p t i o n ) the p o s s i b l e cases of one product molecule . needed to a c t i v a t e a s i t e w i t h j>b = D/(D + L) and <J)L = L/(D + L) and two p r o d u c t - m o l e c u l e s needed to a c t i v a t e a s i t e w i t h <f>ft = D 2/(L + D ) 2 and j>L = 2 2 L /(D + L) are examined. I t i s f u r t h e r assumed t h a t an e x t e r n a l i n f l u e n c e d r i v e s a " f o r c e d c y c l e " by s u p p l y i n g energy t h a t c o n v e r t s L and D back t o A i r r e v e r s i b l y and n o n s t e r e o s p e c i f i c a l l y : Thus f o r m a t i o n r a t e s of enantiomers a r e : (A L J f - kf^R - Kr<t>uL - /TextL 6(0 By s e t t i n g the f o r m a t i o n r a t e s equal to z e r o , the steady s t a t e s both racemic and r e s o l v e d may be o b t a i n e d : - 6 -(a) racemic s t e a d y s t a t e t>= L = RK/LK* (UW (i) In the absence of an e x t e r n a l i n f l u e n c e , the e x p r e s s i o n reduces to 0 - L - H K < / K r r /9/C (s) where K i s the r e c i p r o c a l of the e q u i l i b r i u m c o n s t a n t f o r r e a c t i o n ( 1 ) . (b) r e s o l v e d steady s t a t e s 0=O L = RKf/CKr +KeKt) or (0 L=Q D = RKf/(Kr+Ke«) (f) E q u a t i o n s (6) and (7) apply f o r any model where ( j^ = 0 at D = 0 and s i m i l a r l y f o r tyu . The s t a b i l i t y of steady s t a t e s may be t e s t e d by examining the e f f e c t of a s m a l l d i s t u r b a n c e on the r a t e of change of the f r a c t i o n of D i n the p r o d u c t : cl CD/CD = [L/0> + LfJ $>(k*fl -Krt>) This e q u a t i o n i s a p p l i c a b l e at any p o i n t i n the r e a c t i o n . Now the e f f e c t of s i t e m o d i f i c a t i o n by one and two product m o l e c u l e s on (8) i s examined by s u b s t i t u t i n g the a p p r o p r i a t e f u n c t i o n f o r . The model f o r s i t e a c t i v a -t i o n by one adsorbed p r o d u c t m o l e c u l e i s o b t a i n e d by s u b s t i t u t i n g <|>D = D/(D + L ) , and s i m i l a r l y f o r (j)u i n t o ( 8 ) . i[b/(b*L)]/Jt = CLP/CD+U'J CO - 7 -I f t h e r e i s a r e v e r s e r e a c t i o n i . e . ) 0 , the q u a n t i t y i s n e g a t i v e i f D ^  L and p o s i t i v e i f L ) D. The product always moves toward the racemic steady s t a t e : spontaneous r e s o l u t i o n cannot occur w i t h t h i s model. In the case of s i t e m o d i f i c a t i o n by 2 p r o d u c t mole-c u l e s <j>0 = D 2/(L + D ) 2 and s i m i l a r l y f o r i s sub-s t i t u t e d i n t o (8) to o b t a i n 4Lf>/(D+Lfl/dt = CU>/ro+L)*J^fD-L)Cfl-AcCP-f-L)7 (ie>) C o n s i d e r a d i s t u r b a n c e to the racemic s t a t e w i t h D ^ L or D-L y 0 , then the s i g n of the q u a n t i t y i s t h a t of L~ A-K(D + i^J . I f the e q u i l i b r i u m s t a t e i s the rac e m i c s t a t e , then A = KD = KL and the b r a c k e t e d q u a n t i t y i s n e g a t i v e g i v i n g a s t a b l e racemate. But i f Kext ^ 0 , the racemic s t a t e can become u n s t a b l e . T h i s happens when > K (£> +L) = Z (KrAt) Rkf/(Kr + HKe*t) (u) which reduces t o a comparison of the p r o c e s s e s which r e s t o r e A, Kr < 1 Kext When the e q u i l i b r i u m g r e a t l y f a v o r s the pr o d u c t K f i s s m a l l and any f i n i t e w i l l s u f f i c e to s t a b i l i s e the r e s o l v e d s t a t e . Thus c a t a l y s t upon which product i s ad-sorbed i n the r a t i o .D/L w i l l produce enantiomers i n the 2 2 r a t i o D/L . a m p l i f y i n g any d i s t u r b a n c e from the racemic s t a t e . In summation, the r e a c t i o n must be s t e r e o s p e c i f i c a l l y a u t o c a t a l y s e d and must a l s o be b i m o l e c u l a r i n the product i . e . p r o d u c t f o r m a t i o n r e q u i r e s c a t a l y s t s i t e m o d i f i c a t i o n - 8 -by two product m o l e c u l e s . T h i s may be r e s t a t e d as r e -q u i r i n g a u t o c a t a l y s i s of o r d e r g r e a t e r than one. Thus i f L and D are p r e s e n t on the c a t a l y s t s u r f a c e i n the r a t i o of D/L, the r a t i o . o f f o r m a t i o n r a t e s of D to L 2 2 becomes D /L and a s m a l l i n i t i a l d i f f e r e n c e i n concen-t r a t i o n s i s a m p l i f i e d . A l s o , the system must be examined w e l l b e f o r e e q u i l i b r i u m as r e s o l u t i o n i s p o s s i b l e only away from e q u i l i b r i u m . This p o i n t was not r e c o g n i s e d by p r e v i o u s w o r k e r s . F i n a l l y , the racemic s t a t e i s un-s t a b l e o n l y when product i s removed n o n s t e r e o s p e c i f i c a l l y at a r a t e g r e a t e r than a t h r e s h o l d v a l u e , as g i v e n by H a r r i s o n , which i s a f u n c t i o n of the r e v e r s e r a t e , K ' ' r o n l y . O u t l i n e of E x p e r i m e n t a l Work Isoda's work p r o v i d e d an e x p e r i m e n t a l system i n which some of the parameters had been e x p l o r e d and f o r which some r e s u l t s had a l r e a d y been o b t a i n e d . C o n d i t i o n s s i m i l a r t o those used i n the Japanese work were employed i n t h i s work, but o n l y s l i g h t mention of h y d r o g e n a t i o n times was made i n t h e i r work i m p l y i n g t h a t r e a c t i o n s were pushed to e s s e n t i a l c o m p l e t i o n . In d i s c u s s i n g t h i s work, the t h e s i s i s d i v i d e d i n t o a number of s e c t i o n s which t r e a t i n l o g i c a l o r d e r the d i f f e r e n t a s p e c t s of the e x p e r i m e n t a l work. These s e c t i o n s are o u t l i n e d below so t h a t the g e n e r a l course of the work may be f o l l o w e d e a s i l y . - 9 -1. c<-Ketogl u t a r i c A c i d and " ^ - H y d r o x y g l u t a r i c A c i d . The use o f t h i s p a i r o f compounds by I soda e t . a l . and t h e i r r e p o r t e d spontaneous asymmetry were the d e c i s i v e f a c t o r s i n c hoo s i n g t h i s system f i r s t . S i n c e the r e a c t i o n was mon i t o r ed a t i n t e r m e d i a t e s t a g e s , a q u a n t i t a t i v e method f o r d e t e r m i n i n g h y d r o x y a c i d i n the p re sence o f k e t o a c i d was needed. ' x ' -Hyd roxyg l u t a r i c a c i d i s c o m m e r c i a l l y e x p e n s i v e so s y n t h e s i s from g l u t a m i c a c i d was p e r f o r m e d . M i c r o a n a l y s i s and o p t i c a l r o t a t i o n measurements c o n f i r m e d the i d e n t i t y of the p r o d u c t and measured i t s o p t i c a l p u r i t y . In o r d e r t o a n a l y z e m i x t u r e s of the <x*-hydroxy a c i d and «r -keto a c i d , l ead t e t r a a c e t a t e d e c a r b o x y l a t i o n was per fo rmed i n e x p e c t a t i o n t h a t i t would y i e l d s u c c i n a l d e h y d i c a c i d and s u c c i n i c a c i d . The f i r s t q u a n t i t a t i v e method t r i e d was S c h i f f r e agen t t o de te rm ine the a l d e h y d e . C a l i b r a t i o n cu r ve s made w i t h d i f f e r e n t ba tches of the r eagen t showed poor r e p r o d u c i b i l i t y , so the method was d ropped . The next method t r i e d was quan-t i t a t i v e NMR v i a peak i n t e g r a t i o n . NMR's t aken of the Pb ( 0 A c ) 4 p l u s k e t o a c i d and P b ( 0 A c ) 4 p l u s h y d r o x y a c i d gave i d e n t i c a l NMR's i n d i c a t i n g a d i f f e r e n t r e a c t i o n scheme from the one p r o p o s e d . A f t e r an u n s u c c e s s f u l a t t empt to quan -t i t a t i v e l y p r e c i p i t a t e the k e t o a c i d .wi th 2 , 4 - d i n i t r o p h e n y l -h y d r a z i n e i n advance o f Pb(OAc)^ a d d i t i o n , the d e c a r b o x y l a t i o n approach was d ropped . A method was t r i e d based on the f o r m a t i o n of an h y d r o x y a c i d mo lybdate complex wh ich i s u n a f f e c t e d by the r e d u c t i o n o f the r ema i n i n g mo lybdate to molybdenum b l u e . - 1 0 -The r e d u c t i o n i n molybdenum blue c o l o r from the b l a n k , monitored c o l o r i m e t r i c a l l y , measures the amount of hydroxy-a c i d p r e s e n t . The sour c e r e f e r e n c e i n d i c a t e d t h a t oC-keto-a c i d s d i d not complex w i t h the molybdate, but experiments w i t h o£-ketoglutaric a c i d showed molybdate complexing comparable to t h a t f o r ©('-hydroxyglutaric a c i d . Thus the method was d i s c o n t i n u e d . The l a s t q u a n t i t a t i v e procedure t r i e d was a c i d perman-ganate t i t r a t i o n . T i t r a t i o n r e s u l t s i n d i c a t e d a nonreproduc-i b l e d i f f e r e n c e between the a c i d i f i e d k e t o a c i d and the n e u t r a l i s e d , r e a c i d i f i e d k e t o a c i d which was n e c e s s i t a t e d by the n e u t r a l pH f o r h y d r o g e n a t i o n . NMR's done on the k e t o a c i d and the r e a c i d i f i e d m a t e r i a l were q u i t e d i f f e r e n t . Lack of success i n q u a n t i t a t i v e a n a l y s i s w i t h t h i s system l e d to c o n s i d e r a t i o n of the g l u t a m i c a c i d and oxime system. 2. G l u t a m i c A c i d and o ^ - O x i m i n o g l u t a r i c A c i d . The c i s isomer of the oxime was prepared from < * - k e t o g l u t a r i c a c i d and hy d r o x y l a m i n e h y d r o c h l o r i d e and the m e l t i n g p o i n t checked. The r e l a t i o n between ( + ) - g l u t a m i c a c i d o p t i c a l r o t a t i o n and pH was graphed; the optimum pH f o r r o t a t i o n measurement i s ~ 0 . A s a t i s f a c t o r y q u a n t i t a t i v e method f o r d e t e r m i n i n g g l u t a m i c a c i d i n the presence of oxime was o b t a i n e d w i t h the n i n h y d r i n - amino a c i d r e a c t i o n . C a l i b r a t i o n c u r v e s of absorbance vs ["glutamic a c i d j were prepared u s i n g the c o n c e n t r a t i o n s planned f o r h y d r o g e n a t i o n s . The cu r v e s were s a t i s f a c t o r i l y l i n e a r and r e p r o d u c i b l e . - 11 -3. Raney N i c k e l . A BET s u r f a c e area d e t e r m i n a t i o n was done on the c o m m e r c i a l l y s u p p l i e d c a t a l y s t used i n t h i s work and the r e s u l t s were compared to those o b t a i n e d i n a survey of v a r i o u s raney n i c k e l c a t a l y s t s . The type of c a t a l y s t used by the Japanese workers was found to be more a c t i v e and of d i f f e r e n t s u r f a c e s t r u c t u r e than the commer-c i a l c a t a l y s t . E f f e c t s of s m a l l amounts of 0^ on Ni s u r -f a c e s are mentioned, i m p l y i n g t h a t c a r e s h o u l d be taken to a v o i d exposure of dry R-Ni to 0^ and r e s u l t i n g i n a comparison of the a c t i v i t i e s of wet and dry c a t a l y s t . 4. A d s o r p t i o n S t u d i e s . A d s o r p t i o n experiments of g l u t a m i c a c i d ( G l u ) on Raney n i c k e l s t a r t e d by d e t e r m i n i n g the a d s o r p t i o n of u n n e u t r a l i s e d (pH ~ 3.5) g l u t a m i c a c i d on R-Ni. A s e r i e s of c o n c e n t r a t i o n s was made up and 20 ml of each was added t o .25 g of R-Ni. The d i f f e r e n c e (by n i n h y d r i n ) i n the f i n a l and i n i t i a l G l u c o n c e n t r a t i o n s gave the amount of Glu adsorbed. A d s o r p t i o n r e s u l t s gave a s t r a i g h t l i n e i n a BET p l o t i n d i c a t i n g m u l t i l a y e r adsorp-t i o n . The s u r f a c e area o b t a i n e d was about h a l f the area i n d i c a t e d by the N,, a d s o r p t i o n e x p e r i m e n t . The second experiment d e a l t w i t h Glu a d s o r p t i o n from a s e r i e s of s o l u t i o n s which were i n i t i a l l y e q u i m o l a r i n Glu and phosphate ( P h o s ) . R e s u l t s were much more s c a t t e r e d than those of the f i r s t experiment but i n d i c a t e d c o n s i d e r a b l y reduced Glu a d s o r p t i o n from the p r e v i o u s experiment as w e l l as ev i d e n c e of phosphate i n t e r f e r e n c e i n the n i n h y d r i n r e a c t i o n . - 1 2 -G l u a d s o r p t i o n f r o m e q u i m o l a r m i x t u r e s o f G l u , Phos and oxime (Ox) was e x a m i n e d i n t h e t h i r d e x p e r i m e n t . Re-s u l t s were s i m i l a r t o t h e f i r s t e x p e r i m e n t i n amount o f G l u a d s o r b e d and i n r e p r o d u c i b i l i t y , as c h a r a c t e r i s e d by t h e s m a l l s c a t t e r i n g o f t h e p o i n t s f r o m a f i t t e d c u r v e . The a p p a r e n t p h o s p h a t e i n t e r f e r e n c e w i t h t h e n i n h y d r i n r e a c t i o n r e s u l t e d i n a f o u r t h e x p e r i m e n t where G l u a d s o r p t i o n was s t u d i e d f r o m a s e r i e s o f s o l u t i o n s v a r y i n g i n £Phos) b u t a l l w i t h f G l u ] = 0.001 M. A g r a p h o f G l u a d s o r b e d vs £phos) was U s h a p e d r e v e a l i n g two e f f e c t s : a t low [Vhos] i n t e r f e r e n c e i n t h e n i n h y d r i n r e a c t i o n , l o w e r i n g A as (PhosJ i n c r e a s e s , and c o m p e t i t i o n f o r s p a c e on t h e c a t a l y s t w h i c h t e n d s t o i n c r e a s e A as f P h o s J i n c r e a s e s . S i n c e t y p i c a l r u n c o n d i t i o n s i n v o l v e l a r g e [ P h o s J and low £G 1 u J , s i t e c o m p e t i t i o n i s a l i k e l y f a c t o r i n l i m i t i n g G l u a d s o r p -t i o n and t h e r e f o r e r a t e o f c a t a l y s i s . 5. H y d r o g e n a t i o n Runs. ( a ) O p e r a t i o n o f H y d r o g e n a t o r and P o l a r i m e t e r . The h i g h p r e s s u r e h y d r o g e n a t o r i s d e s c r i b e d m e n t i o n i n g compo-n e n t s , s a f e t y a s p e c t s and o p e r a t i o n . H y d r o g e n a t i o n t e m p e r -a t u r e was l i m i t e d due t o t h e need f o r f r e q u e n t s a m p l i n g and was c o n t r o l l e d by a p r o p o r t i o n a l t e m p e r a t u r e c o n t r o l , t h o u g h t h e s y s t e m d e s i g n made some t e m p e r a t u r e c y c l i n g u n a v o i d a b l e . B e c a u s e o f t y p i c a l l y s m a l l r o t a t i o n s , c l e a n i n g t h e p o l a r i m e t e r c e l l i s i m p o r t a n t . As h y d r o g e n a t i o n s p r o c e e d e d , t h e g r e e n c o l o r o f N i 2 + a p p e a r e d i n s o l u t i o n and when d i l u t e - 1 3 -HC1 was added to the hydrogenated s o l u t i o n , a w h i t e pre-c i p i t a t e r e s u l t e d . R o t a t i o n measurements were only p o s s i b l e by adding more HC1 to d i s s o l v e the p r e c i p i t a t e . D i f f e r e n -t i a t i n g s m a l l r o t a t i o n s from n o i s e was enhanced by measuring r o t a t i o n s at d i f f e r e n t A's and comparing the r e s u l t s to the ORD p a t t e r n f o r g l u t a m i c a c i d . On a graph of o p t i c a l y i e l d vs c h e m i c a l y i e l d ( o f the h y d r o g e n a t i o n ) a h y p e r b o l a was drawn r e p r e s e n t i n g the r e s o l u t i o n of the p o l a r i m e t e r . T h is s e r v e s to i n d i c a t e r e a c t i o n c o o r d i n a t e s i n a c c e s s i b l e to p o l a r i m e t e r measurement i . e . shows the e x p e r i m e n t a l l i m i t a t i o n imposed by low r o t a t i o n s . (b) E v idence of S i d e R e a c t i o n . A r i s e i n pH and the appearance of NH^ as mentioned by the Japanese was c o n f i r m e d . The 2-4 dnph t e s t f o r c ^ - k e t o g l u t a r i c a c i d i n hydrogenated s o l u t i o n was c o m p l i c a t e d by a sl o w e r p a r a l l e l r e a c t i o n of oxime w i t h 2-4 dnph. S i n c e c t f - k e t o -g l u t a r i c a c i d i n s o l u t i o n (though o n l y i n minor amount) i m p l i e s the presence of the r e d u c t i o n p r o d u c t , the hydro-x y a c i d , ORD's of - h y d r o x y g l u t a r i c a c i d and g l u t a m i c a c i d were taken to see i f a s u b s t a n t i a l d i f f e r e n c e e x i s t e d which c o u l d be used to d i s t i n g u i s h them. The ORD p a t t e r n s were q u i t e s i m i l a r , not u s e f u l f o r i d e n t i f i c a t i o n . (c) E x p e r i m e n t a l R e s u l t s . The c o n d i t i o n s used i n the runs are o u t l i n e d as w e l l as the changes made between runs e.g. i n c a t a l y s t b a t c h . Two d i f f e r e n t k i n d s of r a t e law emerged from the 12 e x p e r i m e n t s , l i n e a r and p a r a b o l i c , - 1 4 -o t h e r w i s e the runs, show s i m i l a r i t i e s t h a t make t a b u l a r p r e s e n t a t i o n u s e f u l . Most of the runs produced no o p t i c a l asymmetry, but a few d i d though the r o t a t i o n s i n v o l v e d are very low ( c l o s e to e r r o r l i m i t s ) . Most asymmetric r e -s u l t s are a s s o c i a t e d w i t h runs h a v i n g p a r a b o l i c ( i . e . a u t o c a t a l y t i c ) r a t e laws. Runs doped w i t h ( + ) - g l u t a m i c a c i d d i d not a m p l i f y p r o d u c t i o n of t h a t enantiomer. P o s s i b i l i t i e s f o r f u t u r e work on t h i s system e x i s t both i n a d s o r p t i o n s t u d i e s and i n h y d r o g e n a t i o n k i n e t i c s . E l e c t r o p h o r e t i c m o b i l i t y measurements made of the R-Ni, d e t e r m i n a t i o n of the p o i n t of zero charge of the c a t a l y s t and a d s o r p t i o n b e h a v i o r of Glu w i t h pH would asse s s the e f f e c t of the e l e c t r i c d o u b l e - l a y e r on a d s o r p t i o n . F u r t h e r i n v e s t i g a t i o n of c o m p e t i t i v e a d s o r p t i o n e.g. between Ox and Glu would be u s e f u l . In the h y d r o g e n a t i o n a g r e a t e r range of r e a c t i o n c o n d i t i o n s i . e . temperature and p r e s s u r e might be employed. However, the most c o m p e l l i n g areas of i n v e s t i -g a t i o n are (1) use of more a c t i v e c a t a l y s t e.g. w i t h d i f f e r e n t s u r f a c e s t r u c t u r e and (2) t e s t f o r p o s s i b i l i t y of homogeneous c a t a l y s i s by N i 2 + . Use of the more a c t i v e c a t a l y s t would p e r m i t d u p l i c a t i o n of e x p e r i m e n t a l parameters i n the Japanese (3) work and would a l s o g r e a t l y reduce the time needed f o r a run from a week to a day. The p o s s i b i l i t y of homogeneous c a t a l y s i s v i a N i 2 + i n s o l u t i o n i s of funda-mental importance i n d e t e r m i n i n g r e a c t i o n k i n e t i c s w i t h the - 15 -c o m m e r c i a l l y s u p p l i e d c a t a l y s t used i n t h i s work and t h i s study s h o u l d be pursued i f t h i s c a t a l y s t i s to be used i n f u t u r e h y d r o g e n a t i o n s of the Glu-Ox-Phos system. I t might be u s e f u l to a c q u a i n t the Japanese workers w i t h the r e s u l t s of t h i s study and to d i s c u s s w i t h them p o s s i b l e d i f f e r e n c e s between t h e i r c a t a l y s t and the one used i n t h i s work. A s p e c i f i c p o i n t i n t h i s d i s c u s s i o n might be whether t h e i r c a t a l y s t , l i k e the one used h e r e , showed a tendency t o d i s s o l v e . T his d i s s o l u t i o n p r e -sumably c o n t i n u a l l y renews the a c t i v e s u r f a c e , and might d e s t r o y any tendency towards t e r r i t o r i a l s e p a r a t i o n of enantiomers on the s u r f a c e . - 16 -I I. MATERIALS AND ANALYTICAL METHODS (1) o<-Ketoglutaric Acid and «<-Hydroxyg1utaric Acid Of the two reactant-product pairs studied extensively in (3),the f i r s t studied in th i s work was the o(-ketoglutar ic acid (I) - oc-hydroxyglutaric acid (II) system. I n i t i a l work included synthesis of adequate amounts of the aC-hydroxy acid to permit evaluation of quant itat ive methods for determining the <*-hydroxy acid in the presence of a large amount of the ketoacid. cX-Hydroxyglutaric acid Na s a l t i s prepared from glutamic acid by the method of (9): To 5 g glutamic acid in a 250 ml r. b. f l a sk add 11.5 g NaNO^ dissolved i n d i s t i l l e d water. Place f lask in an ice bath and add 20.8 ml cone. HC1 over 15 minutes (done in a fume hood). Allow NQ^ gas to d iss ipate (^ 1 hr.) and set up reduced pressure apparatus. Boi l o f f c lear solut ion to a mass of white crysta l s (I -carboxy-%-butyrolactone and NaCl) and extract c rys ta l s in a 250 ml beaker with reagent acetone (5 x 40 ml) by vigorous magnetic s t i r r i n g in a fume hood. Concentrated solut ion of the lactone in acetone i s yel low. Each portion of acetone i s allowed to s e t t l e for a few minutes a f ter mixing, then the acetone so lut ion i s f i l t e r e d and added to 30 ml water in a r. b. f lask of reduced pressure apparatus to bo i l o f f acetone. After a l l acetone has been boi led o f f , leaving lactone in aqueous so lut ion, pH i s increased from 2 to 8.5 by adding NaOH so lut ion, hydrolyzing the lactone. The solut ion i s boi led down at t.This section describes unsuccessful attempts to f i nd an ana ly t i ca l method fo r t h i s reaction system. Hydrogenations were done only on the glutamic acid - oxime system; the descr ipt ion of ana ly t i ca l methods for that system begins on p. 53. - 17 -reduced p r e s s u r e to a y e l l o w s t i c k y mass. T h i s i s d i s s -o l v e d i n the minimum amount of hot water and reagent methanol ( /v 1 - 2 ml) i s added. A s m a l l amount of hydroxy-a c i d s a l t p p t s . , but much more p r e c i p i t a t e s when e t h y l e t h e r i s added to the s o l u t i o n . The s a l t comes out e a s i l y at the i n t e r f a c e between the aqueous and e t h e r s o l u t i o n s . The ppt. i s a i r d r i e d and c r u s h e d , washed w i t h reagent methanol then a i r d r i e d and s t o r e d i n a vacuum d e s i c c a t o r f o r /v 1 day. The o(.-hydroxyacid Na s a l t d i d not melt but decomposed at 'v 310 °C and m i c r o a n a l y s i s r e s u l t s were c o n s i s t e n t w i t h the expected weight per cent of C and H: 31.25 and 3.125 % weight expected of C and H r e s p e c t i v e l y , and 31.00 and 3.4-0 % by weight found, a g r e e i n g w i t h i n e x p e r i m e n t a l e r r o r . The o p t i c a l r o t a t i o n of the h y d r o x y a c i d was determined to check the l i n e a r i t y of r o t a t i o n vs. c o n c e n t r a t i o n . The r o t a t i o n of samples of 23%, 38.4-%, 64%, 80% and 100% of an i n i t i a l pH8 b u f f e r e d (2 drops of 0.1M N a ^ O ^ 2 g/ 5 ml h y d r o x y a c i d s o l u t i o n was determined at 27-28 °C u s i n g a 1 cm path c e l l . The r e s u l t s i n d i c a t e d good l i n e a r i t y except at the h i g h e s t c o n c e n t r a t i o n , where the r o t a t i o n was somewhat low ( f i g . 1 ) . Comparison of the product's o p t i c a l r o t a t i o n w i t h t h a t of the pure enantiomer i n d i c a t e s 85% r e t e n t i o n of c o n f i g u r a t i o n from the (-) - g l u t a m i c a c i d s t a r t i n g m a t e r i a l . - 1 8 -Figure 1 <v -Hydroxyglutaric acid Na Salt: Optical Rotation vs. [Hydroxyacid] .40 i -LU o u i Q 365 nm. 436 nm. 546 nm. 578 nm. 589 nm. 100 % stock .042 M [Hydroxyacid] - 1 9 -T a c t i c s f o r D e t e r m i n a t i o n of Sm a l l Amounts of H y d r o x y a c i d i n Large Q u a n t i t i e s of K e t o a c i d The d e t e r m i n a t i o n of s m a l l q u a n t i t i e s of an o^-hydro-x y a c i d i n the presence of an o d - k e t o a c i d can be d i f f i c u l t , due to the s i m i l a r i t y of the ^ - h y d r o x y and o^-keto fu n c -t i o n a l i t i e s . T h e r e f o r e , the f i r s t l i n e of i n v e s t i g a t i o n was to i n c r e a s e the d i f f e r e n t i a t i o n between ( I ) and ( I I ) i n o r d e r t h a t an a p p r o p r i a t e q u a n t i t a t i v e procedure c o u l d more r e a d i l y be found. T h i s was done by t r e a t i n g ( I ) and ( I I ) w i t h Pb(OAc)^. For the h y d r o x y a c i d , HOOC-CH*-CHx-C-CooH + Pk(Oflc)H 1 ™ . Hooc-cHt-CHtrC'c-o-rUoflA tfooc-(ctfJ-cH f HQFlc + P K 0 f l t ) A + cox and s i m i l a r l y , f o r the k e t o a c i d , o HOOC - C H X - C H X - C - COoH HOOC -CHz-CHz.-COOH (V) were the r e a c t i o n s e x p e c t e d . A Warburg apparatus was used to determine the e x t e n t of c o m p l e t i o n of the d e c a r b o x y l a t i o n . The volume of the Warburg apparatus as found by the d e c a r b o x y l a t i o n of a known amount of BaCO^ by HC1, was determined t o be 15.7 ml. - 20 -Lead t e t r a - a c e t a t e d e c a r b o x y l a t i o n of the h y d r o x y a c i d to c o m p l e t i o n would g i v e 9.8 cm Hg p r e s s u r e from 0.0158 g sample at 300° k as compared to 9.5 cm Hg observed e x p e r i m e n t a l l y . Thus e s s e n t i a l l y complete d e c a r b o x y l a t i o n of the h y d r o x y a c i d was i n d i c a t e d . I n i t i a l r e a c t i o n was s w i f t but f i n a l volume was not a c h i e v e d f o r 2-3 h r . Aldehyde D e t e r m i n a t i o n V i a S c h i f f Reagent S i n c e , a f t e r d e c a r b o x y l a t i o n , o n l y aldehyde and a c i d groups are p r e s e n t , a S c h i f f r e agent was used t o q u a n t i t a -t i v e l y determine the aldehyde - and hence the o r i g i n a l h y d r o x y a c i d . A d i r e c t measure of the h y d r o x y a c i d was i m p o r t a n t as i t was expected t h a t l a r g e o p t i c a l y i e l d s might be found e a r l y i n the r e d u c t i o n when o n l y a s m a l l amount of h y d r o x y a c i d was p r e s e n t . The k e t o a c i d would be determined as the d i f f e r e n c e between i n i t i a l k e t o a c i d and c u r r e n t h y d r o x y a c i d . The S c h i f f r e agent used, one of dozens of r e c i p e s i n the l i t e r a t u r e , was made up f o l l o w i n g the sour c e ( 1 0 ) : " F u c h s i n (0.1 g) i s d i s s o l v e d i n 100 cc hot H,,0. The s o l u t i o n i s f i l t e r e d 1 g Na^SO^ and 1 cc c o n c e n t r a t e d HC1 ( s p . g r . 1.18) are added, the r e c e p t a c l e i s put a s i d e f o r 1 h r . , then t r e a t e d w i t h 0.03 g dry a c t i v a t e d C and f i l t e r e d . The f i l t r a t e i s c o l o r l e s s . " - 21 -Kasten (||) i n d i c a t e s t h a t the mechanism of the a l d e -h y d e - S c h i f f r e a c t i o n has been found to vary among alde h y d e s ; a l s o " t h e r e i s no s i m p l e r e l a t i o n s h i p between c o l o r i n t e n -s i t y and aldehyde c o n c e n t r a t i o n " , p a r t l y because more than one dye product may form: R e a c t i o n scheme from (II) p. 44-5 (a) 5 * 0 ^ + H z 0 ^ HZS03 H z50, £± H + + HSOJ (b) H,S0 3 + ZCHO~? ji-l-o-so.H aldehyde b i s u l f i t e a d d i t i o n p roduct ( c o l o r l e s s ) p a r a r o s a n i l i n e ( r e d s o l u t i o n ) so* I f :os p a r a r o s a n i l i n e l e u c o s u l f o n i c a c i d ( c o l o r l e s s ) "s" ~ " O H > \ - y ^ „ y^T S c h i f f s reagent ( c o l o r l e s s ) ^- H N - s u l f i n i c a c i d of p a r a r o -+ ^C5VA/-S0\H s a n i l i n e l e u c o s u l f o n i c a c i d Z«<&^sJ^ ^ O - ^ . ^ double aldehyde s i n g l e aldehyde dye pro d u c t dye product - 22 -Use of l e a d t e t r a a c e t a t e caused a p rob lem when the S c h i f f r e agen t was employed - s o l i d Pb(OAc)^ became s o l u b l e Pb(0Ac)2 a f t e r p e r f o r m i n g d e c a r b o x y l a t i o n but PbSOg p r e c i p i t a t e d out upon a d d i t i o n of S c h i f f r e a g e n t . T h e r e f o r e an e q u i m o l a r amount of py rophosphate {Ua^P^Oj) was added, to remove the l e a d . C a l i b r a t i o n cu r ve s of abso rbance v s . h y d r o x y a c i d c o n c e n t r a t i o n were p repa red u s i n g d i f f e r e n t batches of S c h i f f r e a g e n t , i n o r d e r to e s t a b l i s h the r e p r o d u c i b i l i t y of the method. The f i r s t i t em was to d e t e r m i n e the ^ of maximum a b s o r p t i o n , wh ich depends on the a l d e h y d e . T h i s was found to o c c u r a t 542 nm. The c a l i b r a t i o n p r ocedu re was: d i s s o l v e Q.25 g h y d r o x y a c i d i n 5 m l , put 0.5 ml of t h i s i n a 25 ml v o l u m e t r i c f l a s k w i t h 0.15 g ' P b ( O A c ) ^ , l e t s i t f o r 1 h r , add 0.045 g N a ^ ^ O ^ to o b t a i n w h i t e p p t . , make up to 25 ml w i t h w a t e r , shake and l e t s e t t l e . Draw from the top 0.5 to 0.9 ml of s o l u t i o n and add to two ml S c h i f f r e a g e n t . The r e s u l t s ( f i g . 2) i n d i c a t e d some tendency toward 1 i n e a r i t y but 2 p o i n t s gave A ' s o f 0.9 be l the l i n e sugges ted by the o t h e r p o i n t s . A second ba t ch o f S c h i f f r e agen t was p r epa red u s i n g the same p r o c e d u r e , but t h i s ba t ch t u r n e d out to be much l e s s s e n s i t i v e than the f i r s t b a t c h . With t h i s ba tch 0.8 ml h y d r o x y a c i d s o l u t i o n as made by the p r e c e e d i n g p r e p a r a -t i o n and r e a c t e d w i t h 2 ml S c h i f f gave an A o f o n l y 0.285 c f . A of 1.56 f o r the same h y d r o x y a c i d cone, of the f i r s t - 24 -b a t c h . A c c o r d i n g l y , d i f f e r e n t amounts of h y d r o x y a c i d were used w i t h 2 ml S c h i f f to o b t a i n the optimum maximum A of t\s 1.8. An A of 1.83 was o b t a i n e d w i t h 2.25 ml of the h y d r o x y a c i d s o l u t i o n , and the A from the S c h i f f r e a c t i o n of the same volume of s e v e r a l d i l u t i o n s of the s t o c k h y d r o x y a c i d s o l u t i o n was used to prepare a second c a l i b -r a t i o n curve ( f i g . 3 ) . From the graphs, the r e p r o d u c i -b i l i t y i s poor w i t h the second batch g i v i n g r o u g h l y h a l f the c o l o r of the f i r s t batch f o r the same h y d r o x y a c i d sample. C o l o r development time i n the S c h i f f r e a c t i o n depends on pH and was t y p i c a l l y 2-3 min. f o r these batches w i t h pH 4.0. The n e c e s s i t y of h a v i n g to monitor each sample u n t i l maximum c o l o r was a c h i e v e d (as opposed to a method ha v i n g s m a l l c o l o r l o s s f o r at l e a s t s e v e r a l minutes a f t e r r e a c h -i n g max A very q u i c k l y ) i n a d d i t i o n to the poor r e p r o d u c i -b i l i t y of the method, l e d to abandonment of the S c h i f f method. D e t e r m i n a t i o n of H y d r o x y a c i d by NMR Use of NMR f o r q u a n t i t a t i v e a n a l y s i s of the k e t o a c i d -h y d r o x y a c i d - Pb(OAc)^ system ( r e s u l t i n g i n s u c c i n i c a c i d and s u c c i n a l d e h y d i e a c i d ) depends on (a) the e x i s t e n c e of a s u i t a b l y s t r o n g peak (or peaks) i n the s u c c i n a l d e h y d i c a c i d which i s not s u b j e c t to i n t e r f e r e n c e from o t h e r p r o t o n s o u r c e s i n s o l u t i o n and (b) the c o n t r o l of o p e r a t i n g f a c t o r s a f f e c t i n g q u a n t a t i v i t y such as i n t e g r a l d r i f t and peak s a t u r a t i o n . - 25 -Figure 3 Ca l ib ra t ion of Sch i f f Reagent: A vs. [Hydroxyacid] A [Hydroxyacid] - 26 -NMR's were taken o f I, I I , V, ^ahd! the d e c a r b o x y l a t i o n p r oduc t of II w i t h P b ( O A c ) 4 , ( f i g . 4-7) though o n l y IV and V were e xpec ted a f t e r Pb(OAc)^ t r e a t m e n t . The NMR of the h y d r o x y -a c i d d e c a r b o x y l a t i o n p r oduc t was examined f o r e v i d e n c e of an a ldehyde peak (near 12$ ) but no peak was found t h e r e . I n s t ead a peak a t 2.3$ was found f o r t h a t p r oduc t w h i l e V had one peak a t 2.7& . Any peaks between 4 and 9<£ c o u l d not be ob se rved due . t o the i n t e n s e water peak and i t s s p i n n i n g s i d e b a n d s . The d i f f e r e n c e i n peak p o s i t i o n s of h y d r o x y a c i d d e c a r b o x y l a t i o n p r oduc t and V was c o n f i r m e d by p r e p a r i n g a sample c o n t a i n i n g both ( f i g . 8 ) ; the 0.5 d i f f e r e n c e i n peak p o s i t i o n was m a i n -t a i n e d . Thus a peak from the NMR of the h y d r o x y a c i d d e c a r b o x y -l a t i o n p r o d u c t was s u i t a b l e f o r q u a n t i t a t i v e u se . Assuming the h y d r o x y a c i d d e c a r b o x y l a t i o n p r o d u c t was s u c c i n a 1 d e h y d i c a c i d , q u a n t i t a t i v e d e c a r b o x y l a t i o n a l l o w e d s o l u t i o n s o f known c o n c e n t r a t i o n s to be made up ( v i a the h y d r o x y -a c i d and Pb(OAc)^ method) . I t was found a t the 0.26 M s o l u t i o n used as maximum c o n c e n t r a t i o n t h a t peak s a t u r a t i o n o c c u r r e d : the i n t e g r a l t r a c e f o r 0.26 M was n e a r l y the same as t h a t f o r 0.208 M s u c c i n a l d e h y d i c a c i d . Bejow 0.2 M, i n t e g r a l a m p l i t u d e was l i n e a r w i t h r e s p e c t to c o n c e n t r a t i o n . The q u a n t i t a t i v e a s p e c t of NMR was p r o v i d e d by i n t e g r a t i n g peak a r e a , p r o v i d i n g a measure of the number of s t r u c t u r a l l y i d e n t i c a l p r o t on s p r o d u c i n g the r e s o n a n c e . E r r o r o c c u r r e d because of i n t e g r a l d r i f t i . e . the i n t e g r a l t r a c e tended to r i s e or f a l l over the b a s e l i n e , i n s t e a d of - 27 -Figure 4 N.M.R. Spectrum of -Ketoglutaric acid - 29 -Figure 6 N.M.R. Spectrum of Succinic acid 300 200 1—i—I—I—r 100 ~ i — r Hz i i r T — I — r i — r i. r I 5.0 I l I l I 4.0 3.0 I 1 I 2.0 1.0 £ o - 30 - . Figure 7 N.M.R. Spectrum of Pb(OAc). + -Hydroxyglutaric acid 300 200 100 Hz o Figure 8 N.M.R. Spectrum of Succinic ac id + Succinaldehydic acid - 32 -s t a y i n g c o n s t a n t . I n t e g r a l d r i f t i s more of a problem i f sweep time i s 250 seconds a l t h o u g h s e n s i t i v i t y i s improved - so two s t a n d a r d s e r i e s were done u s i n g 50 and 250 s e c . sweep t i m e s . A l s o , some d i f f e r e n c e was noted i n the s i z e of the i n t e g r a l t r a c e from day to day, u s i n g the same sample, when machine f a c t o r s such as RF power and i n t e g r a l a m p l i t u d e were r e s e t to v a l u e s used p r e v i o u s l y . The sweep time r e f e r s to the p e r i o d used by the machine to scan the o p e r a t i n g range, which i n t h i s case i s 500 Hz. In p r a c t i c e , o n l y the r e g i o n c o v e r i n g the peak of i n t e r e s t i s scanned as the machine can be s e t to scan any p o r t i o n of i t s range. A sample c a l i b r a t i o n curve ( w i t h 50 second I n t e g r a t i o n ) i s reproduced ( f i g . 9 ) . The 250 s e c . I n t . g i v e s an i n t e g r a l t r a c e 3.8 times t h a t of the 50 s e c . I n t . ( f o r a g i v e n c o n c e n t r a t i o n of s u c c i n a l d e h y d i c a c i d ) but i t i s no more l i n e a r . The NMR work to t h i s p o i n t assumed the v a l i d i t y of the d e c a r b o x y l a t i o n r e a c t i o n scheme i n v o l v i n g I , I I , I I I , IV, and V. When the k e t o a c i d and Pb(OAc)^ were mixed, a w h i t e ppt . , p o s s i b l y Pb k e t o g l u t a r a t e , formed i m m e d i a t e l y , b e f o r e CO,, b u b b l e s . No ppt. was observed f o r the hydroxy-a c i d . In o r d e r to study the NMR of s u c c i n i c a c i d u s i n g a sample f r e e of p r e c i p i t a t e , c o m m e r c i a l l y s u p p l i e d s u c c i n i c a c i d was i n i t i a l l y examined d i r e c t l y w i t h o u t u s i n g a sample from the-.ketoacid + Pb(OAc)^ r e a c t i o n . However, when a sample of k e t o a c i d + Pb(OAc),, was made - 33 -Figure 9 N.M.R. Calibration Curve: Relative Peak Area vs. [Hydroxyacid] - 34 -and compared to the h y d r o x y a c i d + PMOAc)^, the NMR's were i n d i s t i n g u i s h a b l e , ( f i g . 10) i . e . both had one peak at 2.3 . As t h i s e v i d e n c e i s i n c o m p a t i b l e w i t h the s i m p l e r e -a c t i o n scheme a l r e a d y proposed, what r e a c t i o n ( s ) causes I and I I to form a p p a r e n t l y the same p r o d u c t ? One p o s s i b i -l i t y i n v o l v e s tautomerism and l a c t o n i s a t i o n : OH HOOC -CHz-CHz~c-c-oH > HoOC~CHrCH=c~c 0 0 ^ . . H O O tautomerism l a c t o n i s a t i o n i ii < ) n O o it o C - C ^ c ^ c f f ^ HOOC-CH^-CH^-CH OH OH One l a s t attempt was made to change the p r o c e d u r e , s t i l l r e t a i n i n g the b a s i c method of Pb(OAc)^ d e c a r b o x y l a -t i o n . P r i o r t o d e c a r b o x y l a t i o n , 2, 4 - d i n i t r o p h e n y l y d r a z i n e was added i n o r d e r to q u a n t i t a t i v e l y p r e c i p i t a t e the k e t o -a c i d : . ... cooH NO± H O O C - CH2'CHX- c-coo// H A / - " \ + o r v - ^ y / V O , \ - 35 -Figure 10 N.M.R. Spectrum of Pb(OAc) + <* -Ketoglutaric acid 300 200 - 36 -G r a v i m e t r i c a n a l y s i s was p e r f o r m e d , u s i n g a r eagen t s o l u -t i o n from S h r i n e r e t . a l . ( 1 2 ) : to 0.8 g 2,4-dnph i n a 50 ml E r l enmeyer i s added 4 ml cone. h^SO^ Water (6 ml) i s added d ropw i se ( w i t h h e a t i n g ) to d i s s o l v e . To warm s o l u t i o n add 20 ml 95 % EtOH. Two k e t o a c i d samples were t e s t e d , a 50 mg sample and a 300 mg.sample; both i n d i c a t e d 95.5% c o m p l e t i o n o f p r e c i p i t -a t i o n . A check of the h y d r o x y a c i d Na s a l t w i t h the 2,4-dnph r eagen t showed e s s e n t i a l l y n i l p r e c i p i t a t i o n . C o n s i d e r i n g t h a t i n t e r e s t i n the r e d u c t i o n r e a c t i o n i s f o cu sed m a i n l y on the r e g i o n of low chem i ca l y i e l d , the p re sence of 5% u n p r e c i p i t a t e d k e t o a c i d was u n d e s i r a b l e . Mo reove r , t he p r e c i p i t a t e was ve r y b u l k y so t h a t the r e a c t i o n of the 2 ,4-dnph r e a g e n t w i t h the c o n c e n t r a t e d k e t o a c i d s o l u t i o n r e s u l t e d , i n e s s e n c e , i n a wet p r e c i p i -t a t e . S i n c e a p r i m a r y conce rn i n NMR i s to e l i m i n a t e any s o l i d p a r t i c l e s i n the sample s o l u t i o n , t h i s was a f u r t h e r d i s advan t ageou s f a c t o r . T h e r e f o r e , i t was d e c i d e d to drop the NMR method of a n a l y s i s as w e l l as the use of P b ( 0 A c ) 4 to d i f f e r e n t i a t e the keto and hydroxy a c i d s to l ook f o r ways of d e t e r m i n i n g h y d r o x y a c i d d i r e c t l y . D e t e r m i n a t i o n o f H y d r o x y a c i d by B l e a c h i n g o f Reduced Mo l ybda te A method f o r the s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of exf - h yd roxy c a r b o x y l i c a c i d s was d e s c r i b e d by M a t u l i s and Guyon (13) and i n v o l v e s f o r m a t i o n of an oi-hydroxyaci d mo lybdate complex which i s u n a f f e c t e d by the r e d u c t i o n o f the r e m a i n i n g f r e e mo lybdate to the c o l o r e d s p e c i e s mo l y -- 37 -bdenum b l u e . In t h e i r work c h l o r o s t a n n o u s a c i d was found the most s u i t a b l e r e d u c t a n t f o r r a p i d i n t e n s e c o l o r development. The b l e a c h i n g e f f e c t on the c o l o r development, as compared to the blank ( w i t h o u t hydroxy-a c i d ) , i s p r o p o r t i o n a l to the amount of h y d r o x y a c i d . M a t u l i s and Guyon suggested t h a t o<-keto a c i d s among ot h e r f u n c t i o n a l i t i e s d i d not b l e a c h i n the same (low) c o n c e n t r a t i o n range as o £ - h y d r o x y a c i d s . Thus, the p o s s i b i l i t y of d i r e c t d e t e r m i n a t i o n of the od-hydro-x y a c i d w i t h o u t i n t e r f e r e n c e by the c<-ketoacid was i n d i c a t e d . The method of (13) was f o l l o w e d , and i s quoted i n p a r t : "Reagents. An a c i d i f i e d s o l u t i o n of sodium molybdate.was prepar e d by adding 25 ml of 1:1 aqueous H^SO^ to a dry 500 ml f l a s k and d i l u t i n g to the mark w i t h 1% sodium molybdate. C h l o r o s t a n n o u s a c i d , 0.85% was p r e p a r e d f r e s h d a i l y by d i s s o l v i n g 1.01 g of F i s h e r Reagent Grade SnCl^.H^O i n 1 ml of c o n c e n t r a t e d HC1 and d i l u t i n g to 100 ml i n a v o l u m e t r i c f l a s k . T h i s c o n t a i n e r must be kept c l o s e d to prevent a i r o x i d a t i o n . " A c a l i b r a t i o n curve f o r the h y d r o x y a c i d was pre-pared by adding 0, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, and 10.0 ml of 0.5% Na h y d r o x y g l u t a r a t e (0.0260M) to 100 ml v o l u m e t r i c f l a s k s . "To each, add 10 ml of the a c i d i f i e d molybdate s o l u t i o n , d i l u t e to 80 ml and mix. S i m u l t a n e o u s l y s t a r t a t i m e r and add 0.5 ml of the - 38 -c h l o r o s t a n n o u s a c i d s o l u t i o n . Q u i c k l y d i l u t e to 100 ml, mix, and read the absorbance a t 5 minutes at 725 nm." P l o t A vs. m i l l i g r a m s of sodium h y d r o x y g l u t a r a t e . The f i r s t h y d r o x y a c i d c a l i b r a t i o n curve ( f i g . 11) had a blank absorbance of ~ 0 . 7 2 but the p o i n t s of the p l o t were b a d l y s c a t t e r e d though the p a t t e r n was s t i l l l i n e a r . In making up the s o l u t i o n s f o r t h i s c a l i b r a t i o n , hand m i x i n g had been used a f t e r a d d i t i o n of c h l o r o s t a n n o u s a c i d . The c a l i b r a t i o n was r e p e a t e d , changing the procedure to employ magnetic s t i r r i n g d u r i n g and a f t e r a d d i t i o n of c h l o r o s t a n n o u s a c i d f o r a s e t p e r i o d (1 min.) i n o r d e r to reduce s c a t t e r i n the r e s u l t s . In a d d i t i o n to r e d u c i n g the s c a t t e r of p o i n t s by a f a c t o r of about 3x, magnetic s t i r r i n g r e s u l t s i n the c a l i b r a t i o n curve l y i n g about 0.09 absorbance u n i t s above the curve produced from the hand mixed samples, but w i t h -out s i g n i f i c a n t l y changing the s l o p e of the l i n e . T h i s i m p l i e s i n c o m p l e t e r e d u c t i o n of molybdate by the c h l o r o -stannous a c i d u s i n g hand m i x i n g , but i t i s not c e r t a i n t h a t a l l f r e e molybdate has been reduced, even u s i n g magnetic s t i r r i n g . Assuming complete r e d u c t i o n of molybdate i n the b l a n k , n, the moles h y d r o x y a c i d / m o l e s 2 -of MoO^ of the complex can be determined and i s found to be 1.74. A = blank absorbance o A = absorbance f o r 50 mg sample A _ A _° - f r a c t i o n of i n i t i a l molybdate i n complex A o Figure 11 Molybdate Blue Calibration: A vs. Weight Hydroxyacid 0.40 I 1 pi r-l r-l 10 '20 1 30 1 40 1 50 °-°0l [Hydroxyacid] 0.002 - 40 -N. = moles h y d r o x y a c i d N m = moles m o l y b d a t e A q = .793 A = .536 N. = .050 - 2.60 x 10~k moli 192 h o n N -4 m .095 = 4.61 x 10 moi 206 es n - 1.74 m o l e s h y d r o x y a c i d / m o l e Mo0^ I n o r d e r f o r t h i s method t o be d e m o n s t r a t e d u s e f u l , i t s t i l l r e m a i n s t o d e t e r m i n e t h e e x t e n t o f b l e a c h i n g w i t h k e t o a c i d . n f o r t h e k e t o a c i d must be much l o w e r ( i d e a l l y 0) t h a n t h e h y d r o x y a c i d n o f 1.7. The s u g g e s t i o n by M a t u l i s and Guyon c o n c e r n i n g low b l e a c h i n g power f o r - k e t o -a c i d s l e d t o t h e e x p e c t a t i o n t h a t n w o u l d be much l e s s t h a n 1 ( ~ 0 . 1 ) . However, when a c a l i b r a t i o n c u r v e u s i n g 0, 2, 5, and 10 ml o f 0.5% Na k e t o g l u t a r a t e was made, ( f i g . 12) i t showed b l e a c h i n g power c o m p a r a b l e t o t h a t o f t h e h y d r o x y a c i d From t h e somewhat s c a t t e r e d p o i n t s o f t h i s c a l i b -r a t i o n c u r v e , an n o f 1.25 was f o u n d . W h i l e i n t h e o r y one c o u l d use t h e method f o r q u a n t i t a t i v e d e t e r m i n a t i o n , g i v e n d i f f e r e n t n v a l u e s , i n f a c t t h e d i f f e r e n c e i s so s m a l l t h a t s e n s i t i v i t y and a c c u r a c y w o u l d be b a d l y i m p a i r e d . T h i s , c o m b i n e d w i t h s c a t t e r i n g on t h e s c a l e o f t h a t i n t h e c a l i b r a t i o n c u r v e p o i n t s , w o u l d c a u s e l a r g e e r r o r s m a k i n g i t an u n a c c e p t a b l e method. - 4 2 -Permanganate T i t r a t i o n The l a s t method examined f o r k e t o a c i d / h y d r o x y a c i d d e t e r m i n a t i o n was a c i d permanganate t i t r a t i o n . Permanganate t i t r a t i o n s must be done i n a c i d (H^SO^) to p r e v e n t an i n t e r -mediate r e d u c t i o n of Mn(VII) to Mn(IV) (as MnC^) from o c c u r r i n g , r a t h e r than the d e s i r e d r e d u c t i o n to M n ( I I ) . A l s o the t i t r a t i o n must be done i n a s u i t a b l e a c i d - one w i t h an an i o n not o x i d i s e d by MnO^. Thus, f o r example, HC1 i s not a s u i t a b l e a c i d . Permanganate i s s t a n d a r d i s e d by t i t r a t i o n w i t h Na o x a l a t e . F r e s h batches of permanganate are made weekly and kept i n dark b o t t l e s to keep the c o n c e n t r a t i o n change due to p h o t o - d e c o m p o s i t i o n of MnO^ to a minimum. The o x i d a t i o n of o x a l a t e by permanganate i s d e s c r i b e d by: 2MnO^~ + 5H 2C 20 Z f —> 2Mn 2 + + 60H" + 2H 20 + 10C0 2 P r e l i m i n a r y s e p a r a t e t i t r a t i o n s of k e t o a c i d and h y d r o x y a c i d were performed. The k e t o a c i d t i t r a t i o n went q u i c k l y a t room te m p e r a t u r e , r e a c h i n g a good c l e a r end-p o i n t , but the h y d r o x y a c i d o x i d a t i o n d i d not occur q u i c k l y at room temp, and even at 60° complete o x i d a t i o n was not o b t a i n e d . From t h i s , i t was c o n c l u d e d t h a t d e t e r m i n a t i o n of k e t o a c i d , w i t h o u t s i g n i f i c a n t o x i d a t i o n of hydroxy-a c i d , c o u l d be ac c o m p l i s h e d by q u i c k room temp, t i t r a t i o n . S i x s o l u t i o n s were made up, each c o n t a i n i n g a con-s t a n t number (1.712 x 10 ) of moles of h y d r o x y a c i d p l u s k e t o a c i d . The s o l u t i o n s c o n t a i n i n g 0, 5, 25, 4-5, 65, and 85% moles h y d r o x y a c i d , were a c i d i f i e d and t i t r a t e d w i t h - 43 -0.01977M MnO^ . The p l o t t e d r e s u l t s ( f i g . 13) of mis. s t a n d a r d i s e d MnO^ vs. % moles h y d r o x y a c i d i n d i c a t e s n e a r l y a s t r a i g h t l i n e . Assuming a two e l e c t r o n o x i d a t i o n of the h y d r o x y a c i d ( i . e . to the k e t o a c i d ) , the o x i d a t i o n of h y d r o x y a c i d i s 10% complete i n the 25 mole % h y d r o x y a c i d sample, d e c r e a s i n g t o 6% i n the 85% hydro-x y a c i d sample. On the b a s i s of the good t i t r a t i o n r e s u l t s , a hydro-g e n a t i o n was t r i e d . Because Raney n i c k e l w i l l d i s s o l v e i n too a c i d s o l u t i o n s , the pH of the i n i t i a l k e t o a c i d s o l u t i o n was r a i s e d t o pH 7.0. When a sample of the r e a c t i o n m i x t u r e was t i t r a t e d , 2.0 times more permangan-ate was used than e x p e c t e d . The i m p l i c a t i o n i s t h a t the n e u t r a l i s a t i o n or subsequent a c i d i f i c a t i o n ( n e c e s s a r y f o r t i t r a t i o n ) changed the k e t o a c i d . A s e r i e s of k e t o a c i d samples was made up to study the e f f e c t s of heat and the order of a d d i t i o n of water and NaOH. H e a t i n g e f f e c t s were s t u d i e d because of the c o n s i d e r a b l e heat r e l e a s e d i n m i x i n g c o n c e n t r a t e d s o l u -t i o n s of k e t o a c i d and base i n the n e u t r a l i s a t i o n f o r the above mentioned h y d r o g e n a t i o n . Two mis. of each of the f o l l o w i n g s o l u t i o n s was t i t r a t e d w i t h 0.01926M MnO^ w i t h an expected amount of MnO^ used of 25.3 ml. MnO^ used (ml) 1 g keto + 1.2 ml H_0 + 10 ml H-0 25.0 a 2 2 1 g keto + 1.2 ml NaOH + 10 ml H.,0 41.4 - 45 -1 g keto + 10 ml H 2 0 + 1.2 ml NaOH + A 1 g keto + 10 ml H 9 0 + 1.2 ml NaOH 32.7 40.2 NMR's of the o r i g i n a l k e t o a c i d , the n e u t r a l i s e d k e t o a c i d and r e a c i d i f i e d k e t o a c i d were taken ( f i g . 1 4 - 1 6 ) . The most p rominent f e a t u r e of the NMR f o r o r i g i n a l k e t o a c i d i s a doub le d o u b l e t w i t h a c e n t e r near 2.8$ and a q u a r t e t c e n t e r e d on 2 . 3 $ . The r a t i o of peak a reas i s ~ 1 . 3 :1 , w i t h the q u a r t e t the s m a l l e r . The Na k e t o g l u t a r a t e NMR was a p a i r o f t r i p l e t s , c e n t e r e d c l o s e t o the q u a r t e t s o f t he o r i g i n a l k e t o a c i d . The r e l a t i v e peak a reas was 1:1.3 w i t h the s m a l l e r peak t h i s t ime be ing d o w n f i e l d ( o p p o s i t e to f i r s t NMR). The NMR f o r the r e a c i d i f i e d k e t o a c i d was the same as t h a t f o r k e t o a c i d w i t h the e x c e p t i o n of a l a r g e peak a t 2.66 tf . The r e l a t i v e peak a reas of the doub le d o u b l e t and the s m a l l q u a r t e t p l u s the new peak was ~ 1 . 3 : 1 , the same r a t i o found f o r the f i r s t NMR. M o l e c u l e s w i t h 2 C=0 bonds s e p a r a t e d by a s i n g l e C-C bond a re s u s c e p t i b l e to eno l f o r m a t i o n , perhaps because of c o n j u g a t i o n . In the case o f o ^ - k e t o g l u t a r i c a c i d : Ho c c CH^-CH^C-OH O o 0 i r HO c - c ^ 'i i o oH - 46 -Figure 14 N.M.R. Spectrum of Unheated <* -Ketoglutaric acid - 47 -Figure 15 N.M.R. Spectrum of Unheated <tf -Ketoglutaric acid Na salt - 48 -Figure 16 N.M.R. Spectrum of the Reacidified Product - 49 -Both forms of the t au tomer i sm e x i s t i n e q u i l i b r i u m , t h e r e f o r e i f the e q u i l i b r i u m l i e s a p p r e c i a b l y toward the eno l f o r m , i t may be d e t e c t e d by the F e C l ^ eno l t e s t . F e r r i c c h l o r i d e s o l -u t i o n was added to s o l u t i o n s o f o r i g i n a l k e t o a c i d , n e u t r a l i s e d k e t o a c i d and the r e a c i d i f i e d f o r m , though HC1 a c i d i f i c a t i o n may a l t e r t e s t r e s u l t s . A c o l o r change ( to red ) was noted f o r the o r i g i n a l k e t o a c i d and f o r the n e u t r a l i s e d k e t o a c i d , but no s u b s t a n t i a l c o l o r change was found f o r the r e a c i d i f i e d samp le . Thus a c i d i f y i n g the Na k e t o g l u t a r a t e d e s t r o y s the e n o l . These r e s u l t s seem to c o n f i r m the unexpec ted r e s u l t o f the mo l yb -date method, wh ich a l s o i n v o l v e d n e u t r a l i s a t i o n to Na k e t o -g l u t a r a t e and a c i d i f i c a t i o n v i a the a c i d i f i e d mo lybdate and c h l o r o s t a n n o u s a c i d s o l u t i o n s . From the eno l t e s t and NMR i t i s c l e a r t h a t the k e t o a c i d has r e a c t e d and f rom the permanganate t i t r a -t i o n s , wh ich v a r i e d o r d e r of a d d i t i o n of base and w a t e r , the change does not seem r e p r o d u c i b l e i . e . i t does not go to c o m p l e t i o n . On the p o s s i b i l i t y t h a t a p o t e n t i o -m e t r i c a l l y f o l l o w e d t i t r a t i o n might y i e l d a c l e a r e r endpo i n t than t h a t a v a i l a b l e v i s u a l l y (by remnant o f MnO^~ c o l o r ) , p o t e n t i o m e t r i c t i t r a t i o n s were pe r fo rmed on Na o x a l a t e , k e t o a c i d and r e a c i d i f i e d k e t o a c i d ( f i g , 17, 18 ) . End p o i n t s a re c l e a r f o r the o x a l a t e s t a n d a r d i s a t i o n t i t r a t i o n and f o r the k e t o a c i d t i t r a t i o n . The e n d p o i n t f o r k e t o a c i d comes 0.3 ml h i g h e r than e x p e c t e d but i s a c c e p t a b l e . However the e n d p o i n t f o r " r e a c i d i f i e d - 52 -k e t o a c i d " i s not d i s t i n c t , the p o t e n t i a l r i s e s g r a d u a l l y over 8 ml. Thus permanganate t i t r a t i o n i s not s u i t a b l e f o r k e t o a c i d / h y d r o x y a c i d d e t e r m i n a t i o n , nor i s any method which g r e a t l y lowers pH. The r e a c i d i f i e d a c i d may be the e n o l l a c t o n e proposed p r e v i o u s l y ( p . 3 V ) . At t h i s p o i n t , the k e t o a c i d / h y d r o x y a c i d system was dropped i n f a v o r of the g l u t a m i c a c i d / k e t o g l u t a r i c a c i d oxime system. - 53 -(2) G l u t a m i c A c i d - -Ox imi nog 1 u t a r i c A c i d System - o x i m i n g l u t a r i c a c i d has c i s - and t r a n s - geo-* m e t r i c i s o m e r s : H00C-C-CH 2 -CH 2 -C00H H00C-C-CH 2 -CH 2 -C00H N 0H t r a n s H0 N c i s J . G. Wood et a l (14) b e l i e v e t h a t the i somer t h a t me l t s 140° i s t he c i s - f o r m , w h i l e the o t h e r wh ich me l t s a t 156 i s the t r a n s - f o rm. Methods of s y n t h e s i s f rom v a r i o u s s t a r t i n g m a t e r i a l s were g i v e n i n (14) f o r both i s o m e r s , but the most c o n v e n i e n t and e f f i c i e n t i n v o l v e d s y n t h e s i s of the c i s - i s ome r . The s y n t h e s i s was c a r r i e d out i n one s tep from < * - k e t o g l u t a r i c a c i d w i t h about 75% y i e l d . Co ld c o n c e n t r a t e d s o l u t i o n s of ©^ - ke tog l u t a r i c a c i d (0.14 mole) and NH 20H.HC1 ( w i t h 10% excess of h y d r o x y l -amine) were u sed . Mas s i ve c r y s t a l l i s a t i o n o c c u r r e d o v e r n i g h t on s t a n d i n g the m i x t u r e i n an i c e b a t h . The c r y s t a l s were c r u shed and a i r d r i e d then s t i r r e d i n 100 ml e t h y l e t h e r f o r an hour t o e x t r a c t u n r e a c t e d k e t o a c i d . The e t h e r was f i l t e r e d o f f and the c r y s t a l s were washed w i t h sma l l p o r t i o n s of c o l d d i s t i l l e d w a t e r , a i r d r i e d and s t o r e d i n a vacuum d e s i c c a t o r . A m e l t i n g p o i n t check o b t a i n e d 1 4 0 ° , i n agreement w i t h ( 1 4 ) . * No ment ion i s made i n (3) which i somer was u sed , nor i s t h e r e any i n d i c a t i o n t h a t two i somers were r e c o g n i s e d . - 54 -" E x p e r i m e n t a l Grade" ( + ) - g l u t a m i c and ( - ) - g l u t a m i c a c i d from F i s h e r Chemicals was used f o r d e t e r m i n i n g o p t i c a l p r o p e r t i e s and f o r t e s t i n g methods of g l u t a m i c a c i d d e t e r m i n a t i o n i n the presence of oxime. I n v e s t i -g a t i o n of o p t i c a l p r o p e r t i e s i n v o l v e d d e t e r m i n i n g de-pendence of r o t a t i o n on pH, to see i f pH s h o u l d be changed from approximate n e u t r a l i t y used i n hydrogena-t i o n . I d e a l l y the pH of the p o l a r i m e t e r sample would be t h a t g i v i n g maximum r o t a t i o n f o r g l u t a m i c a c i d ( i . e . curve maximum) but r o t a t i o n s h o u l d a l s o be n e a r l y i n -dependent of pH over a s u b s t a n t i a l pH r e g i o n ( i . e . broad maximum). S u b s t a n t i a l l y l i n e a r b e h a v i o r of r o t a t i o n vs. c o n c e n t r a t i o n was thought to be a good assumption, e s p e c i a l l y i n the low to moderate c o n c e n t r a t i o n range ( ^ 0.03M) a n t i c i p a t e d to be of most i n t e r e s t i n hydro-g e n a t i o n ( r e d u c t i o n l e s s than 10% c o m p l e t e ) . • Samples of 0.0272 M L - g l u t a m i c a c i d were made up at pH 1.3, 3.33, A-. 4-, 7.95, and 10.0 and r o t a t i o n was measured at 589, 578, 546, 4-36, and 365 nm. The s i z e of r o t a t i o n i n c r e a s e d as o b s e r v i n g A decreased w i t h the r o t a t i o n at 365 nm about 3.5 times t h a t of 589 nm., but r o u g h l y the same r e l a t i o n s h i p i n r o t a t i o n Pi i s main-t a i n e d as pH changes. For L - g l u t a m i c a c i d at low pH t h e r e i s a l a r g e (+) r o t a t i o n t h a t drops q u i c k l y w i t h r i s i n g pH u n t i l around pH5 the r o t a t i o n becomes n e g a t i v e . Maximum c o u n t e r c l o c k w i s e r o t a t i o n o c c u r s around pH 7 - 8, then r o t a t i o n s t a r t s to i n c r e a s e becoming p o s i t i v e around pH 10 ( f i g . 19). Figure 19 Optical Rotation vs. pH at 5 A »s with 0.0272 H Glutamic acid - 56 -S i n c e the pH range from 1.5 to 10 d i d not any-where i n d i c a t e a h i g h r o t a t i o n w i t h a s m a l l (d r o t / d pH ), samples of lower pH were examined. G l u t a m i c a c i d samples of 0.1361 M were made up w i t h pH's of "v -.48, ^> - 0.30, ~ - 0.18, 0, 0.3, 1.3, 1.7, 2.2, 2.8. Samples of pH } 0 were made up by adding 6 M HC1 to 20 ml of 0.340 M g l u t a m i c a c i d s t o c k s o l u t i o n u n t i l the r e q u i r e d pH was reached and making up to 50.0 ml t o t a l volume w i t h d i s -t i l l e d water and 6 M HC1 t o h o l d the pH c o n s t a n t . Monit-o r i n g v i a pH meter was o n l y p o s s i b l e i n the no n n e g a t i v e pH r e g i o n , so f o r pH < 0 t h e . g l u t a m i c a c i d s o l u t i o n was assumed to a c t o n l y as a d i l u t a n t on the 6 M HC1. The most i m p o r t a n t f e a t u r e of the pH dependence of r o t a t i o n i n t h i s r e g i o n i s the f l a t n e s s of the curve ( f o r a l l /\ *s) between pH ^ - 0 . 5 and +.8 ( f i g . 2 0 ) . Thus r o t a t i o n i s l a r g e and pH dependence i s minimal as d e s i r e d at low pH ( ^ 0 ) . Comparison of £ <>C J ^ e x p e r i m e n t a l and l i t e r a t u r e v a l u e (15) i s r e a s o n a b l e , but not e x a c t . With r = .0595°, n / v = 20 g / 1000 c c , 1 = 0.1 dm, [ J p 5 = 29.8 c f 32.0 = C 3 D 3 1 A t ' v a l u e - Another source (16) g i v e s £ ^ J ^ = 30.3° i . e . not a l l s o u r c e s agree, but temperature d i f f e r e n c e i s almost c e r t a i n l y the main reason f o r the d i f f e r e n c e , as the s i z e ( ^ 5% i n 5°) i s t y p i c a l ( 1 7 ) . D e t e r m i n a t i o n of G l u t a m i c A c i d by N i n h y d r i n Method The n i n h y d r i n r e a c t i o n : Figure 20 - 57 -Optical Rotation vs. pH at 5 ^  's with 0.1361 M Glutamic acid 0.220 0.210 + 0.190 deg. 0.170 0.150 0.130 0.110 0.090 0.070 0.050 h 0.030 J L -0.5 1 I I I I 0.5 1.0 1.5 2.0 2.5 P H 3.0 - 58 -- o • o i s the c l a s s i c a l method f o r d e t e r m i n i n g amino a c i d s , where (X) i s the c o l o r e d ( b l u e ) s p e c i e s monitored s p e c t r o -p h o t o m e t r i c a l l y at 570 nm. The n i n h y d r i n method of T r o l l and Cannin (18) i n v o l v e s s u b s t a n t i a l use of nonaqueous s o l u t i o n s to e f f e c t n e a r l y the maximum p o s s i b l e c o l o r development. P o t a s s i u m c y a n i d e i s used as a r e d u c i n g agent to s u p p l y the n e c e s s a r y reduced form of n i n h y d r i n , h y d r i n d a n t i n . P y r i d i n e s e r v e d as a b u f f e r but a l s o , w i t h a p h e n o l - a l c o h o l s o l u t i o n , i n c r e a s e d both r a t e and y i e l d of the n i n h y d r i n r e a c t i o n , so t h a t c l o s e to 100% of t h e o r e t i c a l c o l o r was o b t a i n e d . The reagents as used here were as i n ( 1 8 ) : " 1. N i n h y d r i n s o l u t i o n . 500 mg of n i n h y d r i n are d i s s o l v e d i n 10 ml of a b s o l u t e a l c o h o l . 2. 80 per cent phenol s o l u t i o n . 80 g of reagent grade phenol are d i s s o l v e d i n 20 ml of a b s o l u t e a l c o h o l , w i t h g e n t l e h e a t i n g . The s o l u t i o n i s shaken w i t h 1 g of P e r m u t i t f o r about 20 minutes to remove t r a c e s of ammonia and then decanted. - 59 -3. K C N - p y r i d i n e r e a g e n t . 2 ml of a 0.01 M s o l u t i o n of KCN a re d i l u t e d to 100 ml w i t h ammon ia - f ree p y r i d i n e , p r epa red by s h a k i n g 100 ml of p y r i d i n e w i t h 1 g of P e r m u t i t f o r about 20 m i n u t e s . 4. 60 per c e n t a l c o h o l (by v o l ume ) . " P r o cedu re - 0.5 ml o f aqueous g l u t a m i c a c i d s o l u t i o n (o f any pH between 1 and 8 ) , c o n t a i n i n g up to 1.0 jumoles o f amino a c i d , i s heated w i t h 1 ml of K C N - p y r i d i n e r e a g e n t and 1 ml of 80 per c e n t phenol r e a g e n t i n a 10 ml v o l u m e t r i c f l a s k i n a b o i l i n g wa te r b a t h . When the m i x t u r e has r eached the t e m p e r a t u r e of the wate r b a t h , 0.30 ml of n i n h y d r i n s o l u t i o n i s added, the 10 ml f l a s k i s s t o p p e r e d and the r e a c t i o n i s a l l o w e d t o p roceed f o r 5 m i n u t e s . The s o l u t i o n i s c o o l e d and made up to 10 ml w i t h 60 per c e n t a l c o h o l and-the o p t i c a l d e n s i t y a t 570 nm i s d e t e r m i n e d . One h a l f ml of d i s t i l l e d w a t e r , s u b j e c t e d to the same p r o c e d u r e i s used as a r e a g e n t b l a n k . The q u a l i t y of n i n h y d r i n used i s i m p o r t a n t w i t h r e s p e c t t o the r e p r o d u c i b i l i t y of r e s u l t s . The e x p e r i e n c e i n t h i s work was t h a t M a l i n c k r o d t Reagent Grade n i n h y d r i n gave q u i t e r e p r o d u c i b l e r e s u l t s w i t h an abso rbance sp read of about * 0.01 f o r samples of the same • s o l u t i o n . A good n i n h y d r i n r e agen t ( i . e . r eagen t 1) has a f a i n t green c o l o r and g i v e s a l i g h t g reen c o l o r e d b l a n k . The o t h e r s ou r ce o f n i n h y d r i n was MCB Reagent n i n h y d r i n wh i ch p roduced a n i n h y d r i n r e a g e n t hav ing a y e l l o w i s h orange c o l o r . T h i s r e agen t gave b l u e b l a n k s , w i t h the c o l o r r e s u l t i n g not from wate r but from - 60 -p y r i d i n e . The r e p r o d u c i b i l i t y was much reduced; the absorbance spread f o r samples of the same s o l u t i o n i n -c r e a s e d to + (0.03 - 0.05). Two c a l i b r a t i o n curves were p r e p a r e d , based on the need to be a b l e to monitor a c c u r a t e l y at e a r l y s t a g e s of r e a c t i o n (0 - 10%'< c o m p l e t i o n ) as w e l l as throughout the r e d u c t i o n (0 - 100% c o m p l e t i o n ) . A c o n c e n t r a t e d s o l u -t i o n of 2 g oxime / 20 ml (.6211 M) i s the s o l u t i o n used i n the h y d r o g e n a t i o n . With samples of 10% and 100% c o m p l e t i o n t h i s g i v e s 0.06211 M and 0.6211 M g l u t a m i c a c i d r e s p e c t i v e l y . By d i l u t i n g the f i r s t 35x and the second 350x the s o l u t i o n s are reduced to 1.76 - 3 - 7 x 10 M whence 0.5 ml c o n t a i n s 8.87 x 10 mole g l u t -amic a c i d . Two s e t s of s o l u t i o n s were made up: one w i t h t o t a l moles of g l u t a m i c a c i d and oxime of 8.80 x 10 ^ moles - 6 and the o t h e r w i t h 8.80 x 10 moles. In the f i r s t s e t , s o l u t i o n s of 0, 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% t o t a l moles g l u t a m i c a c i d were made up from s o l u t i o n s of n e u t r a l i s e d 0.00176 M g l u t a m i c a c i d and oxime. The r e s u l t s of the n i n h y d r i n procedure on the s o l u t i o n s are g i v e n i n f i g . ( 2 1 ) . In the second s e t , s o l u t i o n s of 0, 2, 4, 6, 8, and 10% t o t a l moles were prepared from s o l u t i o n s of n e u t r a l i s e d .0176 M g l u t a m i c a c i d and oxime ( f i g . 2 2 ) . In both cases "0%" t o t a l moles g l u t a m i c a c i d does not mean water but 0.00176 M and 0.0176 - 61 -Figure 21 Calibration Curve for Ninhydrin Reaction: A vs. ft Moles Glutamic acid (0 - 100) <) . I • I . I • i • i 0 20 40 60 80 100 % moles Glutamic acid - 63 -M oxime, so t h a t some a d s o r p t i o n ( ~ 0.1) i s a t t r i b u -t a b l e to oxime. Both curves e x h i b i t good l i n e a r i t y and as they were p l o t t e d A v s . % moles g l u t a m i c a c i d (not M), they can be used to f i n d the c h e m i c a l y i e l d of a h y d r o g e n a t i o n d i r e c t l y . - 64 -(3) Raney N i c k e l : C h a r a c t e r i s t i c s and Hand l i n g Raney N i c k e l ( R -N i ) does not r e f e r to a u n i f o r m sub-s tance w i t h a s i n g u l a r s e t of p r o p e r t i e s but to a range of m a t e r i a l s produced by p roce s se s a l l i n v o l v i n g l e a c h i n g of N i - A l a l l o y w i t h NaOH to produce a c h e m i c a l l y a c t i v e s u r f a c e on wh ich c o n s i d e r a b l e amounts of H a re a d s o r b e d . R-Ni a c t i v i t y v a r i e s a c c o r d i n g to c o n d i t i o n s of p r e p a r a -t i o n and, e s p e c i a l l y f o r more a c t i v e c a t a l y s t s , dec rea se s s u b s t a n t i a l l y ove r t ime (days or week s ) . A l s o c a r e must be taken to i s o l a t e the R-Ni from a t m o s p h e r i c as a low p a r t i a l p r e s s u r e of oxygen i s s u f f i c i e n t t o form a mono layer of NiO t he r eby g r e a t l y r e d u c i n g c a t a l y t i c e f f e c t . To reduce the l i k e l i h o o d of i n a d v e r t a n t exposu re to a i r , R-Ni i s s h i pped and s t o r e d i n a can under w a t e r . The R-Ni used i n t h i s work was o b t a i n e d f rom W. R. Grace & Co. (a d i v i s i o n of Dav i son Chemica l Co.) i n one pound packages and was t h e i r No. 28 Raney A c t i v e N i c k e l C a t a l y s t . T h i s c a t a l y s t was produced by the l e a c h i n g w i t h a l k a l i o f 50% Ni - 50% A l a l l o y powder but pa rameter s o f t e m p e r a t u r e and t ime a re not s p e c i f i c a l l y known. In o r d e r t h a t a d s o r p t i o n on the R-Ni c o u l d be d i s c u s s e d somewhat q u a n t i t a t i v e l y , a BET s u r f a c e a rea d e t e r m i n a t i o n was made on a 2.586 g sample of R -N i . The f i r s t s t ep i n the a rea d e t e r m i n a t i o n was f i n d i n g the volume of t h a t p o r -t i o n of the vacuum system c o n t a i n i n g the R-Ni s t a r t i n g from the known volume of a s e c t i o n of the vacuum system t h a t i s - 65 connected by sto p c o c k 4 to the unknown volume vacuum R-Ni For t h i s volume d e t e r m i n a t i o n He i s used s i n c e He adsorbs n e g l i g i b l y on R-Ni. Hence a change i n p r e s s u r e i n the known volume ( V ^ ) , o b t a i n e d by opening stopcock 4 and a l l o w i n g the He access to the unknown volume (v.,), i s due e n t i r e l y t o volume change, not a d s o r p t i o n . I n t o tube T (from which s t o p c o c k 5 had been removed) was added wet R-Ni. 5 was r e i n s e r t e d and the tube was opened to vacuum. C a r e f u l h e a t i n g w i t h a hot a i r gun e x p e d i t e s removal of water w i t h o u t bumping. A c o l d t r a p must be used downline t o keep water out of the pump. When an a p p r o p r i a t e vacuum was been a t t a i n e d ( /-v 10 ^ t o r r ) 1 i s c l o s e d and the manometer i s zer o e d . A d s o r p t i o n proceeds t o a g r e a t e r e x t e n t at low temp e r a t u r e , t h e r e f o r e the sample c e l l i s immersed i n l i q u i d N^. He i s a d m i t t e d by opening 2 w i t h a l l oth e r s t o p c o c k s c l o s e d , then 2 i s c l o s e d and the - 66 -p r e s s u r e i s measured as w e l l as temp e r a t u r e . F i n a l l y 4 and 5 are opened and p r e s s u r e and temperature measurements are ag a i n made. The c y c l e - v a c u u m - P ^ ^ 2 ^ 2 was re p e a t e d to o b t a i n 4 s e t s of r e a d i n g s . With V-^  = 63.4 ml, a = 43.0 ml was found. In the a d s o r p t i o n experiment i s added c u m u l a t i v e l y i . e . a s m a l l i n i t i a l i s used ( ~ 6 cm'Hg),4 i s opened then c l o s e d a f t e r i s measured, and the next increment of i s added to o b t a i n the next P^. i s s l i g h t l y a f f e c t e d by p r e s s u r e change s i n c e empty space i n the manometer forms p a r t of i t . Hence a c o r r e c t i o n term of the form ( v o l / cm Hg.) i s i n c l u d e d . F i g . (23) shows moles adsorbed v s . e q u i l i b r i u m p r e s s u r e ( i n atm.). Below 0.01 atm. moles adsorbed i n -cre a s e s very q u i c k l y w i t h p r e s s u r e but approaches a con-s t a n t much lower s l o p e above .01 atm. The BET e q u a t i o n X = 1 + ( C - 1 ) X V ( l - X) cv CV m m where X = P . , / 76.0 = p r e s s u r e i n atm. eq u i 1 . r V = moles adsorbed V = moles adsorbed i n a monolayer m J C = c o n s t a n t was used i n a computer program to f i t the best s t r a i g h t l i n e from a p l o t of X / V ( l - X) vs. X so t h a t V M and C c o u l d be found from i n t e r c e p t and s l o p e ( f i g . 2 4). The p l o t i s q u i t e l i n e a r w i t h = 1.264 x 10"^ moles / mono-- 69 -l a y e r and C = 90.811. Hence assuming the area / molecule of N £ i s 16.2 A 2, area is 123.3 m2 or 47.7 m2 / g R-Ni. F r e e l et a l (1^) determined s u r f a c e areas of R-Ni samples produced from d i f f e r e n t p r o c e d u r e s , as w e l l as samples from Davison Chemicals Co. t h a t i n c l u d e d a c a t a l y s t made from 50 wt. % Ni which i s p r o b a b l y (though t h i s i s not e x p l i c i t l y s t a t e d ) t h e i r product No. 28, the same used i n t h i s work. They r e p o r t c o n s i d e r a b l y l a r g e r areas ( ^ 80 2 m /g) w i t h no s u b s t a n t i a l area change from weeks to months i n water s t o r a g e . Chemical a n a l y s i s of the Davison Co. samples i n d i c a t e d 91 - 96 wt % N i and 3 - 8 wt. % A l --more l e a c h i n g than any of the o t h e r samples a n a l y s e d which had 80 - 90 wt % N i or l e s s . The e x t e n t of l e a c h i n g and p r o b a b l y the c o n d i t i o n s i n v o l v e d e.g. temperature a f f e c t the c a t a l y s t s u r f a c e a r e a . Isoda (3) et a l used the R-Ni and r e p o r t s i n l a t e r work (20) t h a t R-Ni used i n t h a t work was " s i m i l a r to t h a t des-c r i b e d " i n (3) "One and a h a l f gms of a powdered R-Ni a l l o y ( N i : A l = 40:60) was added to 20 ml of 20% sodium h y d r o x i d e S o l u t i o n i n s m a l l p o r t i o n s d u r i n g 5 minutes and was a l l o w e d to s t a n d f o r 45 minutes at 80° C. The n i c k e l c a t a l y s t was washed s e v e r a l times w i t h water". The E n g l i s h t r a n s l a t i o n of (3) c o n t a i n s no p r e p a r a t i v e i n f o r m a t i o n on the R-Ni used. Using p r e p a r a t i v e methods s i m i l a r to t h a t i n (XO), 2 F r e e l et a l o b t a i n e d BET areas of 95 m /g though a c o n s i d e -- 70 -r a t i o n of work done i n (IH) on mean pore s i z e may be more r e l e v a n t to c a t a l y t i c a c t i v i t y and mechanism. F r e e l et a l found t h a t the commercial c a t a l y s t s n o r m a l l y p r e p a r e d under severe c o n d i t i o n s (b. p. of aqueous a l k a l i s o l u t i o n ) to have mean pore d i a m e t e r s (based on pore volume d e t e r m i n a t i o n ) more than t w i c e t h a t of W,- type c a t a l y s t ( rsj 62 /-v 25 %) but t h a t i f the more a c t i v e , l e s s l e a c h e d sample i s s u b j e c t e d to f u r t h e r l e a c h i n g , a h i g h pore volume s t r u c t u r e r e s u l t s . Pore s i z e s were found not to be a c o n t i n u o u s d i s t r i b u t i o n but m a i n l y of 2 s i z e ranges. Thus W,- c a t a l y s t c o n t a i n s a preponderance of s m a l l e r pores w h i l e the commercial pre-p a r a t i o n has the l a r g e r . D i s s i m i l a r i t y of s u r f a c e s t r u c t u r e s may r e s u l t i n changes i n the mechanism of h y d r o g e n a t i o n t h a t would l e a d to d i f f e r e n c e s i n a b i l i t y to produce an asymmetric p r o d u c t . As the s u r f a c e i s very s u s c e p t i b l e t o c o m b i n a t i o n w i t h oxygen, care must be taken t h a t d r i e d R-Ni i s not exposed to a i r , nor even to L grade which c o n t a i n s ^ 1 % 0^. Work by Norton and Tapping ( 2 1 ) , i n d i c a t e s t h a t o n l y a very low exposure ( ^ 10 ^ t o r r s) of 0^ i s needed to form a monolayer on a Ni s u r f a c e and t h a t much h i g h e r exposure i s needed to produce any f u r t h e r change. Though the s u r f a c e s are not the same ( N i and R - N i ) , the i n d i c a t i o n i s c l e a r t h a t 0^ be a v o i d e d , even i n minute amounts. Normal l y t h i s i s not a problem as R-Ni i s p r e p a r e d i n aqueous s o l u t i o n and used i m m e d i a t e l y . In t h i s work, - 71 however, i t was d e s i r e d to dry i t so t h a t the weight used would be a c c u r a t e l y known. A dry box c o n t i n u o u s l y f l u s h e d w i t h G grade N^, gas (low 0^ c o n t e n t ) , was used to h o l d a d e s i c c a t o r i n which was kept the d r i e d R-Ni. The R-Ni was removed from the de s i ' c c a t o r and handled i n the dry box where p o r t i o n s were added to a screw top v i a l used f o r c a r r y i n g R-Ni to an e l e c t r i c b a l a n c e . A check was made of the a c t i v i t y of the d r i e d R-Ni vs the wet m a t e r i a l , by comparing h y d r o g e n a t i o n r a t e s under i d e n t i c a l r e a c t i o n c o n d i t i o n s ( d e t a i l s i n the Hydrogenation Runs s e c t i o n ) . Three samples of s l u r r y were weighed and d r i e d , then reweighed. The % wt of R-Ni i n the s l u r r y was c o n s t a n t to + 1.6 % at 57.7 % by w e i g h t . The r e a c t i o n r a t e and shape of r e a c t i o n c u r v e (% chem y i e l d v s . time) were very s i m i l a r . Thus o x i d a t i o n i n the d r i e d R-Ni i s minor i n e x t e n t or e f f e c t . - 72 -I I I . ADSORPTION STUDIES ON RANEY NICKEL The k i n e t i c s of Raney N i c k e l r e d u c t i o n of the oxime to g l u t a m i c a c i d a r e l i k e l y to be dependent on t h e s t r e n g t h of a d s o r p t i o n of r e a c t a n t , p r o d u c t and b u f f e r . F u r t h e r m o r e t h e y may depend on t h e r e l a t i v e s t r e n g t h of a d s o r p t i o n , so f o r example, i f the p h o s p h a t e b u f f e r a d s o r b e d p r e f e r e n t i a l l y to t h e oxime or g l u t a m i c a c i d i t would s e r i o u s l y i n t e r f e r e w i t h c a t a l y s i s . The a d s o r p t i o n o f g l u t a m i c a c i d ( G l u ) on R-Ni was t h e f i r s t a d s o r p t i o n e x p e r i m e n t p e r f o r m e d . A BET p l o t f r o m t h e s e d a t a e s t a b l i s h e d N , the number of moles i n a mono-m l a y e r w h i c h , combined w i t h an e s t i m a t e of t h e a r e a c o v e r e d by one G l u m o l e c u l e , gave an a r e a f o r R-Ni w h i c h c o u l d be compared to the N^ a d s o r p t i o n r e s u l t s . The e f f e c t o f p h o s p h a t e and oxime on G l u a d s o r p t i o n was s t u d i e d by d e t e r m i n i n g G l u a d s o r p t i o n f i r s t i n an e q u i m o l a r m i x t u r e o f G l u and p h o s p h a t e (Phos) and t h e n i n a m i x t u r e of G l u , Phos, and oxime (Ox). In the i n i t i a l e x p e r i m e n t u s i n g o n l y G l u as a d s o r b a t e , a s e r i e s o f G l u s o l u t i o n s r a n g i n g from 0.050 M to 0.001 M was made ;up. Samples of 0.250 g R-Ni were weighed out and added to 20 ml of each of t h e s o l u t i o n s . G l u was d i s s o l v e d and used d i r e c t l y w i t h o u t n e u t r a l i s a t i o n by NaOH, hence t h e pH was m i l d l y a c i d 3.5. A d s o r p t i o n i s c h a r a c t e r -i s t i c a l l y t e m p e r a t u r e d e p e n d e n t , so t h a t t e m p e r a t u r e - 73 -m o n i t o r i n g , i f not c o n t r o l , was n e c e s s a r y . I t was found t h a t the l a b was, though not good, s a t i s f a c t o r y i n main-t a i n i n g the same temp. (23° t 1°). A f t e r two days of s h a k i n g i n corked 50 ml erlenmeyer f l a s k s , samples of the s o l u t i o n s were taken and d i l u t e d to a maximum concen-t r a t i o n of 0.001 M ( i f no a d s o r p t i o n ) . Samples of the i n i t i a l s o l u t i o n s were d i l u t e d to determine Glu v i a the absorbance from the n i n h y d r i n r e a c t i o n . The method of T r o l l and Cannon (17) was used to o b t a i n a number of 0.001 M s o l u t i o n s as b l a n k s . R e s u l t s of the blank sam-p l e s and the adsorbed samples are i n c l u d e d to i l l u s t r a t e an a d s o r p t i o n experiment by.example. O r i g i n a l F i n a l A ( B l a n k ) A(adsorbed s o l u t i o n ) S o l u t i o n Cone. 0.05 M 0.001 M 1.11 1.03 .965 1.05 0.025 " 1.12 1.005 1.04 1.02 0.01 " 1.13 .915 .965 .96 0.005 " 1.14 .83 .82 0.0025 " 1.14 .74 .75 0.001 " .61 A ( b l a n k ) i s the absorbance expected i f no a d s o r p t i o n o c c u r s ; i t s v a l u e i s 1.13. For each of the f i r s t t h r e e s o l u t i o n s , o n l y the two c l o s e s t v a l u e s of A were used i n d e t e r m i n i n g an average. Then the grams Glu adsorbed was c a l c u l a t e d : ( A ( b l a n k ) - A( adsorbed ))/CGlu}. . . ,1(MW)(volume)=g. A ( b l a n k ) V i n i t i a l / A e.g. ((1.13 - 1.04)/1.13)(0.05)(147)(0.020)= .0117 = - 74 -From t h i s , X = g G l u a d s o r b e d / g R-Ni = s i n c e 0.250 g R-Ni was u s e d . The e q u i l i b r i u m c o n c e n t r a t i o n o f G l u i s g i v e n by fGluJ e q u i l = ( A ( b l a n k ) - A ( a d s o r b e d ) ) / A ( b l a n k C G l u i n i t I t i s i n t e r e s t i n g t o compare t h e c o r r e s p o n d e n c e o f t h e r e -s u l t s t o t h e BET and L a n g m u i r m o d e l s . The BET e q u a t i o n ( 1) models m u l t i l a y e r a d s o r p t i o n w h i l e t h e L a n g m u i r e q u a t i o n (2) assumes o n l y m o n o l a y e r a d s o r p t i o n . BET X(P P ) e X C m (C - 1) P, X C P m o L a n g m u i r (1) (2) B X m X X r B C e q u i l i b r i u m c o n e , o f G l u s a t u r a t e d c o n e , o f G l u - 0.054 M ( a t 23°) g G l u a d s o r b e d / g R-Ni g G l u c o n s t a n t c o n s t a n t i n m o n o l a y e r / g R-Ni [G l u J i n i t . A X (g) P • ! (M) e q u i l P /X e P IP e o P e / X ( P 0.05 1 . 04 0 .0468 0 .0460 0 .983 0.852 123 0.025 1. 02 0 .0286 0 .0226 0.790 0.419 25.2 0.01 0 . 96 0 .01768 0 .00850 0 .481 0 .157 10.57 0.005 0 . 83 0 .0156 0 .00367 0.235 0 .0680 4.67 0.0025 0 . 75 0 .00988 0 .00166 0.168 0 .0307 3 . 21 0.001 0 . 61 0 .0054 0 .00054 0.100 0.010 1 . 87 A p l o t o f 9A vs P e f i g • ( 2 5 ) shows a r a p i d i n c r e a s e i n f o r P ^ 0. 005, b u t i n d i c a t e s l i n e a r i n c r e a s e f o r h i g h e r P. - 75 -Figure 25 Glutamic acid Adsorption Isotherms on Raney Nickel: g. Glu adsorbed vs. [Glu] for 0.25 g. R-Ni. 5 $ Reaction i n Hydrogenation 0.014 0.012 h 0.002 0.01 0 0 2 [Glu] 0 0 3 eq. 0.04 0.05 - 76 -The Langmuir t e s t p l o t , P g/X vs P g was l i n e a r i n the r e g i o n P g 0.009 ( f i g . 26) but d e v i a t e d c o n s i d e r a b l y at h i g h e r c o n c e n t r a t i o n . However the BET p l o t ( f i g . 27') was l i n e a r to P g / P q > 0.4 which i s i n good agreement w i t h the s t a n d a r d P g / P Q range a p p l i c a b l e to a d s o r p t i o n t h a t f o l l o w s the BET model v i z . m u l t i l a y e r a d s o r p t i o n . From the p l o t , w i t h i n t e r c e p t = 1.263 and s l o p e = 57.26, X^ = 1.709 * 1 0 " 2 g Glu / monolayer. Assuming an area f o r Glu of 40 $ 2 / m o l e c u l e , S, the s u r f a c e area covered by a monolayer can be found: - ? n p S = X N A ' 1 0 'IM = 28.0 i r r / g . m m 3 T h i s area i s about h a l f t h a t found by N,, a d s o r p t i o n BET, which may not d i s c r e d i t the e a r l i e r r e s u l t s but may r a t h e r r e f l e c t c h e m i s o r p t i o n onto s i t e s . The second experiment examined Glu a d s o r p t i o n onto R-Ni from an e q u i m o l a r m i x t u r e of Glu and Phos, a d j u s t e d to pH - 6.0 w i t h NaOH. The same procedure and range of s o l u t i o n s was used i n each e x p e r i m e n t . The range of A v a l u e s f o r the b l a n k s i n t h i s e xperiment proved to be l a r g e r than i n the f i r s t experiment w i t h A = 1.06 - 2.5%. Because of the l a r g e v a r i a t i o n i n A v a l u e s and the expec-t a t i o n t h a t phosphate i n t e r f e r e n c e i n the n i n h y d r i n r e -a c t i o n would lower A v a l u e s , an A - 1.08 was chosen which was a v a l u e i n c l u d e d i n the range of A's o b s e r v e d . I t i s c l e a r t h a t the range of A v a l u e s i n the blank w i l l i n t r o -- 79 -duce a l a r g e amount of e r r o r i n the a d s o r p t i o n at h i g h c o n c e n t r a t i o n s i n c e r e l a t i v e l y s m a l l changes i n c o n c e n t r a -t i o n are i n v o l v e d . This i s r e f l e c t e d i n the l a r g e e r r o r b a rs at l a r g e f G l u ) i n the p l o t of X v s . C G l u l ( f i g . 25) which i s a l s o a f u n c t i o n of v a r i a t i o n i n the A v a l u e s of r e p e a t e d n i n h y d r i n s done on each adsorbed s o l u t i o n . Because of the l a r g e e r r o r l i m i t s the e x p e r i -ment was r e p e a t e d and f o u r n i n h y d r i n Glu d e t e r m i n a t i o n s were done on each s o l u t i o n . The r e p r o d u c i b i l i t y of the second s e t of data d e t e r i o r a t e d from t h a t of the f i r s t s e t , w i t h v a r i a t i o n s i n A commonly of 5%. Comparison of the graphs i n d i c a t e s t h a t the e x t e n t of a d s o r p t i o n of Glu by i t s e l f and i n the presence of Phos i s c o n s i d e r a b l y d i f f e r e n t , though l a r g e e r r o r i s i n v o l v e d i n the Phos + Glu e x p e r i m e n t . Phosphate appears to i n t e r f e r e s e r i o u s l y i n the a d s o r p t i o n of G l u , r e d u c i n g i t by 4x a t h i g h c o n c e n t r a t i o n . The t h i r d e x p e r i m e n t , i n v o l v i n g the Glu a d s o r p t i o n from an e q u i m o l a r m i x t u r e of G l u , Phos and Ox a d j u s t e d to pH = 6.0, gave r e s u l t s of c o n s i d e r a b l y improved r e p r o d u c i b i l i t y over the Glu + Phos exp e r i m e n t . The v a r i a t i o n i n blank A v a l u e s was - 2% -- t h i s l e d to g r e a t l y reduced e r r o r bars i n the a d s o r p t i o n i s o t h e r m f o r t h i s experiment ( f i g . 2 5 ) . A l s o , the s c a t t e r i n g of the p o i n t s from a f i t t e d curve was much reduced here over the p r e v i o u s e x p e r i m e n t . - 80 -I t i s i n t e r e s t i n g to note t h a t the e x t e n t of Glu a d s o r p t i o n i n the Glu-Phos-Ox experiment was about the same as t h a t i n the g l u expe r i m e n t , a t h i g h c o n c e n t r a t i o n . . The d i s c r e p a n c y i n the a d s o r p t i o n i s o t h e r m of the second experiment w i t h those of the f i r s t and t h i r d experiments may r e s u l t from a c t u a l d i f f e r e n c e s i n a d s o r p t i o n , or p a r t i c u l a r l y i n the second e x p e r i m e n t , from l a r g e e r r o r s . The s u p p r e s s i o n of Glu a d s o r p t i o n by Phos i s e x p l i c a b l e i n e i t h e r of two ways: (a) c o m p e t i t i v e a d s o r p t i o n of the two s p e c i e s on the same s i t e s ; (b) the b u f f e r i n g a c t i o n of the Phos, which changes the pH from ^ 3.5 ( G l u s o l u t i o n o n l y ) to 6.0 ( a l l s o l u t i o n s c o n t a i n i n g P h o s ) . Thus the Glu s p e c i e s adsorbed from Glu + Phos s o l u t i o n s i s a n i o n i c w h i l e t h a t adsorbed from G lu s o l u t i o n s w i t h o u t Phos i s the n e u t r a l m o l e c u l e . The enhancing e f f e c t of Ox on Glu a d s o r p t i o n i s u n u s u a l . I t i s n o t i c e a b l e o n l y above 25% s u r f a c e coverage and i n c r e a s e s r a p i d l y as 100% coverage i s reached and passed. Hence the e f f e c t of Ox may be mainly on adsorbed Glu l a y e r s beyond the f i r s t and may be produced by Ox i n the f i r s t l a y e r a d s o r b i n g Glu on top of i t s e l f more s t r o n g l y than does Glu i t s e l f , or by s t r o n g Glu - Ox i n t e r a c t i o n s i n the l a y e r s beyond the f i r s t s t a b i l i s i n g the mixed system. T h i s might a r i s e from s t r o n g e r hydrogen bonding between an NH,, and an NOH than between two NH^'s or two NOH's. - 81 -The e r o s i o n of r e p r o d u c i b i l i t y and the d e c l i n e of the A blank v a l u e i n the second experiment as compared to the f i r s t experiment suggested t h a t the e f f e c t of phosphate s h o u l d be examined as a s e p a r a t e v a r i a b l e . An a d s o r p t i o n experiment was done u s i n g a range of phosphate c o n c e n t r a -t i o n s (from 0.001 to 0.05 M as b e f o r e ) a l l i n a 0.001 M Glu s o l u t i o n . The e f f e c t of i n c r e a s i n g phosphate concen-t r a t i o n on A blank v a l u e s ( f i g . 28) s u p p o r t s the i d e a of phosphate i n t e r f e r e n c e i n the n i n h y d r i n r e a c t i o n . In f i g . ( 2 8 ) , the A v a l u e f o r Phos = 0 came from the A blank v a l u e of 1.13 from the f i r s t e x p e r i m e n t . The a d s o r p t i o n r e s u l t s based on the A v a l u e s of the n i n h y d r i n r e a c t i o n would be expected t o r e f l e c t the c o n c e n t r a t i o n dependence of the phosphate i n t e r f e r e n c e w i t h the n i n h y d r i n r e a c t i o n . I f t h i s was the o n l y f a c t o r a f f e c t i n g A, o t h e r than a c o n s t a n t a d s o r p t i o n of Glu independent of [ P h o s ] , the a d s o r p t i o n curve one would expect would be the same shape as f i g . (28) but s h i f t e d u n i f o r m l y i n A to r e f l e c t Glu a d s o r p t i o n . Another p o s s i b l e e f f e c t i s c o m p e t i t i o n between Phos and Glu f o r space on the c a t a l y s t . T his would mean decreased a d s o r p t i o n of Glu at high CPhos}; i f t h i s were the o n l y f a c t o r a f f e c t i n g absorbance, A would i n c r e a s e w i t h CPhosO. The absorbance r e s u l t s of the a d s o r p t i o n experiment show t h a t both e f f e c t s are i n v o l v e d -- the e f f e c t of Phos vs Glu s i t e c o m p e t i t i o n at h i g h [ P h o ^ f o r m i n g a U shaped curve from what would o t h e r w i s e have been a curve of - 82 -Figure 28 Effect of [Phosphate] on Ninhydrin Reaction of 0.001 M Glu: Ninhydrin A vs. [Phosphate] - 83 -n e g a t i v e s l o p e ( f i g . 29) caused by the p redominant e f f e c t of phosphate i n t e r f e r e n c e w i t h the n i n h y d r i n r e a c t i o n . The e f f e c t demons t ra ted i n f i g . (29) does not nec -e s s i t a t e a l a r g e c o r r e c t i o n i n the abso rbance v a l u e s of the Phos + Glu a d s o r p t i o n e x p e r i m e n t . Large [Phos ] s t r o n g l y a f f e c t s the s m a l l amount o f G lu p r e s e n t i n the f i n a l a d s o r p t i o n e x p e r i m e n t , but e q u i m o l a r c o n c e n t r a t i o n s of Phos and Glu i n the second expe r imen t r e s u l t i n l i t t l e change e v i d e n t i n abso rbance v a l u e s . The r e s u l t from the a d s o r p t i o n s tudy most s - i g n i f i c a n t to the whole work was e v i d e n c e of s t r o n g a d s o r p t i o n . In f i g . (25) the Glu i s o t h e r m c r o s s e s the X m = 0.00425 g l i n e ( de t e rm ined f rom BET) s i g n i f y i n g c o m p l e t i o n o f the mono l a ye r . T h i s o c c u r s a t 0.006 M G lu o r 1% c h e m i c a l y i e l d i n the h y d r o g e n a t i o n . The l a r g e i n i t i a l c o n c e n t r a t i o n s of Phos and Ox w i l l r e s u l t i n a c o m p l i c a t e d , c o m p e t i t i v e , m u l t i l a y e r a d s o r p t i o n . A l l of the a d s o r p t i o n e x p e r i m e n t s e xcep t the f i r s t were per fo rmed a t the pH used i n the h y d r o g e n a t i o n s , 6 .0 . At t h i s pH the g l u t ama te a n i o n , not the n e u t r a l atom, i s the s p e c i e s ad so rbed - - thus a more s o p h i s t i c a t e d model than BET i s needed to a ccoun t f o r cha rge e f f e c t s . At o p e r a -t i o n a l pH an e l e c t r i c a l doub le l a y e r i s formed which a f f e c t s a d s o r p t i o n b e h a v i o r and which i s pH dependent . For Glu a d s o r p t i o n a t pH 3 .5 , n e u t r a l m o l e c u l e s were adsorbed and the i s o t h e r m f o l l o w e d the BET model w e l l . - 85" -In a more comp le te s tudy of t h i s s y s t e m , the p o i n t of ze ro charge o f R-Ni c o u l d be de te rm ined and e l e c t r o -p h o r e t i c m o b i l i t y measurements taken to de te rm ine the e f f e c t of pH on a d s o r p t i o n . - 86 -Hyd rogena t i on R e s u l t s of the G l u t a m i c A c i d Oxime System - 87 -IV. HYDROGENATION RUNS O p e r a t i o n o f H y d r o g e n a t o r and P o l a r i m e t e r P r o p e r o p e r a t i o n of t h e h i g h p r e s s u r e h y d r o g e n a t o r ( A m e r i c a n I n s t r u m e n t Co., S i l v e r s p r i n g Md., C a t . No. 40-12155 SP) i s q u i t e s i m p l e but v e r y i m p o r t a n t i n d e t e r -m i n i n g t h a t t h e r e s e a r c h e r w i l l be on hand to f i n i s h h i s r e s e a r c h . The h i g h p r e s s u r e h y d r o g e n a t o r on::'the r o o f of t h e C h e m i s t r y b u i l d i n g i s t h e r e s p o n s i b i l i t y o f Dr. R o s e n t h a l -- he and h i s s t u d e n t s were v e r y h e l p f u l i n d e m o n s t r a t i n g i t s o p e r a t i o n and i n s u p p l y i n g n e c e s s a r y p a r t s . The f o l l o w i n g a c c o u n t i s p r e s e n t e d i n s u f f i c i e n t d e t a i l to i n f o r m f u t u r e u s e r s of t h e same a p p a r a t u s . Any s y s t e m m o d i f i c a t i o n s such as c h a n g i n g r u p t u r e d i s c s i n t h e b l o w - o u t a s s e m b l y to p e r m i t h i g h e r p r e s s u r e s s h o u l d be c h e c k e d by l o o k i n g i n t h e company c a t a l o g u e to e n s u r e t h a t ill J. components a r e r a t e d f o r more s e v e r e c o n d i t i o n s . A l l f i t t i n g s used must be c o m p a t i b l e to t h e s y s t e m i n t h r e a d and maximum p r e s s u r e s p e c i f i c a t i o n . The h i g h p r e s s u r e h y d r o g e n a t o r s c h e m a t i c a l l y d e p i c t e d i n f i g . (30) c o n s i s t s o f : 1. a h y d r o g e n t a n k w i t h v a l v e VI i n t h e top c o n n e c t e d to a s t a i n l e s s s t e e l t u b e l e a d i n g t o the s h a k e r h e a t e r a s s e m b l y 8. As an o p t i o n a l b y p a s s to t h e n o r m a l gas r o u t e , 2. i s a gas f i l t e r w h i c h was not used i n t h i s work. Thus V2 was a l w a y s c l o s e d . When H£ p r e s s u r e i n 1. i s l o w e r t h a n t h a t r e q u i r e d f o r h y d r o -g e n a t i o n t h e c o m p r e s s o r 3. i s used t o r e a c h t h e d e s i r e d Figure 30 Schematic Diagram of High Pressure Hydrogenator - 89 -p r e s s u r e . The compressor motor i s c o n t r o l l e d by a s w i t c h on the west w a l l of the c o n t r o l room. Valve V4 c o n t r o l s the primary gas r e l e a s e which runs the gas o u t s i d e through s t e e l t u b i n g and t h e r e i s an a l t e r n a t e r e l e a s e c o n t r o l l e d by V7. The exhaust l i n e h ere, however, i s only tygon and a too qui c k p r e s s u r e r e l e a s e w i l l pop the l i n e from i t s j o i n w i t h V7. Gauge Gl i n d i c a t e s h y d r o g e n a t i o n p r e s s u r e when i n t e r -v ening v a l v e s V5 and V6 between Gl and r e a c t i o n bomb 7. are open. The gauge i s i n the h y d r o g e n a t i o n room and can be seen through a window from the c o n t r o l room. The t u b i n g s p i r a l s l o o s e l y i n t o 4. which i s a h o r i z o n t a l p i v o t a l l o w i n g the shaker mechanism to move i n a v e r t i c a l p l a n e . The s p i r a l adapts the r i g i d tube from the non-moving to the r o c k i n g frame. The r u p t u r e d i s c ' a s s e m b l y 6. i s a s a f e t y f e a t u r e to l i m i t p r e s s u r e w e l l below r a t e d p r e s s u r e t o l e r -ances of the o t h e r elements of the system. A U shaped tube connects 6. to the bomb when the bomb i s se a t e d i n the s h a k e r - h e a t e r assembly 6. The shaker mechanism 5. c o n s i s t s of a motor which d r i v e s a s e t of l i n k a g e s con-nected t o the bomb w e l l and i s c o n t r o l l e d by a s w i t c h i n the c o n t r o l room. The e x c u r s i o n of the l o n g i t u d i n a l shaker a x i s i s from /v 50° to 10° to the h o r i z o n t a l . The r e a c t i o n v e s s e l or bomb, see f i g . ( 3 1 ) , i s a t h i c k w a l l e d s t e e l c y l i n d e r 30 cm l o n g and 6 cm i n diameter w e i g h i n g A/ 10 kg. I t has an o u t s i d e t h r e a d around the top and a s e a l i n g r i n g on the top which matches t h a t on the - 90 -Figure 31 Schematic Diagram (Cross-section) of High Pressure Hydrogenator Reaction Vessel 11 a h o s 6 - 91 -s e a l e r p l a t e 2. Th i s p l a t e has a threaded h o l e i n the middle which i s the gas i n l e t and which c o u p l e s t o the U shaped tube. Over t h i s f i t s a s t e e l cap 3. which t h r e a d s onto the bomb. E i g h t t h r u s t b o l t s threaded through the cap are t i g h t e n e d w i t h a t o r q u e wrench to a c h i e v e a s e a l by p r e s s i n g the p l a t e 2. down. The e i g h t b o l t s l i e e q u a l l y spaced i n a c i r c l e over the s e a l i n g r i n g and i f the b o l t s are numbered c l o c k w i s e 1 to 8, the o r d e r of t i g h t e n i n g and l o o s e n i n g s h o u l d f o l l o w the symmetry of 1-5-3-7-6-2-4-8. When t i g h t e n i n g the t h r u s t b o l t s the bomb i s h e l d i n a sta n d b u i l t f o r i t l o c a t e d i n the c o n t r o l room. A l l the b o l t s are t i g h t e n e d f i r s t to 20 l b . , then to 50 l b . w i t h the t o r q u e wrench. To a v o i d s h e a r i n g o l d b o l t s which may f r e e z e i n the cap, i t i s i m p o r t a n t t o l u b r i c a t e the t h r u s t b o l t s . T h i s i s best done w i t h g r a p h i t e l u b r i c a n t . The v e s s e l c o n t a i n i n g the r e a c t a n t s o l u t i o n i s a pyrex l i n e r w i t h an o.d. a couple of mm l e s s than the i . d . of the bomb. Enough f l a t metal s p r i n g s 5. are p l a c e d i n the bottom of the bomb so t h a t the top of the l i n e r shows ^> 1 cm above the top of the bomb. The t e n s i o n caused by t i g h t e n i n g the cap keeps the l i n e r from moving i n d e p e n d e n t l y The l i n e r cap and l i n e r are connected w i t h a ground g l a s s j o i n t which must be l i g h t l y greased to keep the cap from s t i c k i n g or f r e e z i n g to the l i n e r . - 92 -A s m a l l h o l e halfway a l o n g the pyrex l i n e r a l l o w s gas p r e s s u r e to e q u a l i z e q u i c k l y . S i n c e the bomb i s t i l t e d i n s h a k i n g , the l i n e r must be o r i e n t e d w i t h the hole up. Proper o r i e n t a t i o n of the l i n e r i n the bomb can be as s u r e d b e f o r e the bomb i s f i t t e d i n the w e l l because the bomb has on l y one c o r r e c t o r i e n t a t i o n i n the w e l l . A s m a l l h o l e i n the bottom of the bomb s e a t s a complementary peg p r o j e c t i n g from the w e l l bottom. Hy d r o g e n a t i o n p r o c e d u r e ; The capped l i n e r c o n t a i n -i n g r e a c t a n t s o l u t i o n ( u s u a l l y 20 ml) i s i n s e r t e d i n t o the bomb and o r i e n t e d . The p r e s s u r e p l a t e and cap are put on and the cap i s th r e a d e d t o hand t i g h t n e s s . T h i s i s c o n v e n i e n t l y done i n the bomb h o l d e r i n the c o n t r o l room. The t h r u s t b o l t s are t i g h t e n e d i n two st a g e s w i t h a t o r q u e wrench t o 20 and 50 l b . i n a 1-5-3-7-6-2-4-8 type p a t t e r n . The t i g h t e n e d bomb i s c a r r i e d to the h y d r o g e n a t i o n room and s e a t e d i n the bomb w e l l . The U shaped c o n n e c t o r i s i n s t a l l e d t i g h t e n i n g each end i n s t a g e s , a l t e r n a t e l y . A f t e r c h e c k i n g a l l c o n n e c t i o n s f o r t i g h t n e s s , check t h a t V2, V4 and V7 are c l o s e d . Open V5 and V6 l e a v i n g VI and V3 c l o s e d . S l o w l y open V I , then open V3 s l i g h t l y and watch gauge Gl through the window. When Gl i n d i c a t e s 200 l b . c l o s e V3 and open V4 a l l o w i n g gas to escape. This f l u s h i n g procedure removes most of the a i r from the ap p a r a t u s ; c l o s i n g V4 and r e p e a t i n g the procedure t w i c e - 93 -more removes almost a l l a i r . A f t e r f l u s h i n g , V3 shut, G l i s watched f o r a minute t o see i f p r e s s u r e f a l l s n o t i c e -a b l y from a l e a k . The system i s t e s t e d f o r slow l e a k s downline of V6 by dabbing j o i n t s w i t h soap s o l u t i o n . Leaks were o c c a s i o n a l l y found at e i t h e r end of the U shaped c o n n e c t o r and were s e a l e d by f u r t h e r t i g h t e n i n g w i t h a wrench. L a s t l y V I , V3, V5 and V6 are c l o s e d and the shaker motor i s t u r n e d on. Because of temperature e f f e c t s on a d s o r p t i o n and r e -a c t i o n r a t e and perhaps mechanism, tem p e r a t u r e m o n i t o r i n g and c o n t r o l are i m p o r t a n t . In (3) the r e d u c t i o n s were g e n e r a l l y done at e l e v a t e d temperature and the e x p e r i -ments which r e s u l t e d i n the l a r g e s t o p t i c a l r o t a t i o n were done at 80°. In t h i s work h y d r o g e n a t i o n s were not done at temperatures h i g h e r than 30-35° and o f t e n l o w e r . Low t e m p e r a t u r e s i n t h i s work f a c i l i t a t e d the f r e q u e n t s a m p l i n g n e c e s s a r y f o r k i n e t i c s t u d i e s . I n i t i a l l a c k of s u i t a b l e temperature c o n t r o l equipment r e s u l t e d i n the f i r s t h y d r o g e n a t i o n s b e i n g done at room temperature w i t h the t emperature measured by a thermometer which, v i a a hole i n the bomb j a c k e t , was i n d i r e c t c o n t a c t w i t h the bottom of the bomb. Temperature c o n t r o l was a c h i e v e d w i t h a h e a t e r d r i v e n by a p r o p o r t i o n a l temperature c o n t r o l ( p . t . c . ) which was connected to a t h e r m i s t o r i n s t a l l e d i n the thermometer w e l l . Because of the d i s t a n c e of the h e a t e r t o the t h e r m i s t o r (the h e a t e r c o i l s were halfway up the bomb) t h e r e was "the r m a l - 94 -o v e r r u n " which kept the bomb temperature h i g h e r than t h a t s e t on the p . t . c . When the t h e r m i s t o r caused the p . t . c . to reduce or shut o f f c u r r e n t to the h e a t e r , t h e r e was a thermal g r a d i e n t from the t h e r m i s t o r t o the h e a t e r t h a t caused an o v e r r u n . A c a l i b r a t i o n curve was pre-pared f o r the F i s h e r . p . t . c . ( f i g . 32) and the appro-p r i a t e s e t t i n g made f o r the d e s i r e d t e m p e r a t u r e , 30°. Thermal ov e r r u n was reduced by i n t r o d u c i n g a V a r i a c p o t e n t i o m e t e r between the h e a t e r and p . t . c . and s e t t i n g the V a r i a c at 60 v o l t which was the minimum v o l t a g e needed to o p e r a t e the h e a t e r . The therm a l g r a d i e n t r e s u l t e d i n about ± 2° temperature v a r i a t i o n from c y c l i n g . A d e t a i l e d account of the P e r k i n Elmer 141 P o l a r i -meter i s a v a i l a b l e i n the o p e r a t i n g manual and so i s not r e p r o d u c e d , but some p r o c e d u r a l p o i n t s s h o u l d be e x p l a i n e d . Due to the s m a l l s i z e of r o t a t i o n s , c l e a n -l i n e s s of the p o l a r i m e t e r c e l l i s i m p o r t a n t . The 1 cm c e l l i s f i l l e d w i t h chromic s u l f u r i c a c i d and l e f t f o r s e v e r a l minutes, then s u c t i o n d r i e d and s t r o n g l y r i n s e d w i t h a j e t of d i s t i l l e d w ater. To a v o i d l o c a l h e a t i n g e f f e c t s from a c i d - w a t e r m i x i n g the c e l l i s s u c t i o n e d dry both b e f o r e and a f t e r a d d i t i o n of a c i d w i t h a p i p e t t e . The c e l l i s c l e a n e d b e f o r e each run and r measurements were made of the empty c e l l t o compensate f o r c e l l e f f e c t s . As d i s c u s s e d p r e v i o u s l y , the o p t i c a l r o t a t i o n i s measured at pH 0. Th i s i s a l t e r e d from the h y d r o g e n a t i o n - 96 -pH of 6 by the a d d i t i o n of an e q u a l amount (0.55 ml) of d i l u t e HC1 and thorough m i x i n g . In h y d r o g e n a t i o n s which were c a r r i e d to a s i g n i f i c a n t l e v e l of c o m p l e t i o n ( ( 10 % ) , a w h i t e p r e c i p i t a t e was found to form on the a d d i t i o n of a c i d which d i s s o l v e d on m i x i n g . At h i g h e r l e v e l s of c h e m i c a l y i e l d , the ppt. d i d not c o m p l e t e l y d i s s o l v e u n t i l more HC1 was added. S i n c e the course of h y d r o g e n a t i o n i s p a r a l l e l e d by the appearance of d i s s o l v e d Ni i n s o l u t i o n a N i 2 + c o o r d i n a t i o n compound may be i n v o l v e d . P r e c i p i t a t e or bubbles i n the p o l a r i m e t e r c e l l or temperature change d u r i n g measurement can a l t e r r e s u l t s . S o l i d p a r t i c l e s , e i t h e r the w h i t e ppt. or c o l l o i d a l R-Ni from the c a t a l y s t , are d i s s o l v e d on s u f f i c i e n t a d d i t i o n of HC1. Bubbles o f t e n form a t c e l l ends and are removed by t i l t i n g the c e l l and t a p p i n g to cause the bubble to r i s e to a sample i n l e t h o l e and escape. Due to the e n c l o s e d p o l a r i m e t e r h o l d i n g a r e a , i n s i d e the machine, and s i n c e the machine was always t u r n e d on h a l f an hour b e f o r e measurement, a l l o w i n g temperature e q u i l i b r i u m to be reached, a n e a r l y c o n s t a n t measurement temperature of 26-28° was o b t a i n e d . E r r o r s i n s i n g l e measurements can be avoided by t a k i n g r e a d i n g s at a l l a v a i l a b l e ~A 's of the p o l a r i m e t e r . S i n g l e /\ measurements are o n l y r e p r e s e n t a t i v e of the o p t i c a l r o t a t o r y d i s p e r s i o n (ORD) c u r v e , but i f measure-ments at s e v e r a l d i f f e r e n t /\ 's are made, the r e s u l t s can be checked f o r correspondence t o the expected r e l a t i o n -s h i p as g i v e n by ORD. Thus the magnitude of the r o t a t i o n - 97 -i n d i c a t e s an o p t i c a l l y a c t i v e s u b s t a n c e , w h i l e the p a t t e r n of r vs A a f f i r m s t h a t the o p t i c a l l y a c t i v e m a t e r i a l i s c o n s i s t e n t w i t h g l u t a m i c a c i d . S i n c e t h i s work i n v o l v e s m o n i t o r i n g o p t i c a l asymmetry ( " o p t i c a l y i e l d " ) s t a r t i n g at very low product c o n c e n t r a t i o n ("chemical y i e l d " ) i t i s u s e f u l to examine the l i m i t a t i o n imposed by a p o l a r i m e t e r s e n s i t i v i t y of 0.001° on a c c e s s i b i l i t y of measurement. Using the r e l a t i o n s h i p fo^J = r v and the T T T e x p e r i m e n t a l parameters, the a c c e s s i b i l i t y of r e a c t i o n c o o r d i n a t e s (chem. y i e l d , o p t i c a l y i e l d ) t o o p t i c a l measurement can be e s t a b l i s h e d by s e t t i n g a minimum s i g n i f i c a n t r v a l u e . With C^Jp = 31.4°, a c e l l p a t h l e n g t h of 1 dm and a maximum g l u t a m i c a c i d c o n c e n t r a t i o n i n the c e l l of 0 . 3 f O 6 M, the per cent o p t i c a l r e s o l u t i o n P nec e s s a r y f o r p o l a r i m e t e r d e t e c t i o n w i t h r = 0.002° can be found as a f u n c t i o n of the per cent c h e m i c a l y i e l d X: P ={0.002)(1000)(2)(1002) = 13.95 (31.4)(147)(0.6211) X X From the graph of the h y p e r b o l a ( f i g . 3 3 ) , the l i m i t a -t i o n s i n the d e t e c t a b i l i t y of the v a r i o u s p o s s i b l e c o n d i t i o n s of c h e m i c a l and o p t i c a l y i e l d i s e v i d e n t . For i n s t a n c e i f ch e m i c a l y i e l d i s 1 %, then no s i g n i f i c a n t r o t a t i o n w i l l r e s u l t u n l e s s the product i s at l e a s t 15 % o p t i c a l l y r e s o l v e d . The graph i s s i g n i f i c a n t i n i t s r e l a t i o n to the o b j e c t i v e of f i n d i n g h i g h o p t i c a l p u r i t y e a r l y i n the r e d u c t i o n . I f " e a r l y " means l e s s than 1 % c h e m i c a l y i e l d , the t e c h n i q u e s used i n t h i s work c o u l d not determine o p t i c a l r e s o l u t i o n . For - 99 -X > 1 %, modest P v a l u e s (5 to 10 %) can be determined, a l t h o u g h f o r s m a l l r o t a t i o n s ( r ^ 0.005) the r e l a t i v e e r r o r i s l a r g e . H a n d l i n g of Samples to A v o i d Hold Growth In some e a r l y runs samples o c c a s i o n a l l y grew mold because of e x c e s s i v e time between s a m p l i n g and a d d i t i o n of HC1. The m a t e r i a l c o u l d not be seen b e f o r e the a d d i t i o n of a c i d , but a f t e r w a r d s appeared as w h i t e h a i r l i k e s t r a n d s . A s o l u t i o n c o n t a i n i n g g l u t a m i c a c i d and phosphate b u f f e r e d to pH 6 might be expected t o d i s p l a y s u i t a b i l i t y as a growth medium f o r a i r - b o r n e organisms. D i f f e r e n t methods of con-t r o l were i n v e s t i g a t e d : (a) h a n d l i n g samples under a l o n g wave UV lamp; (b) t r y i n g to f i n d a g e r m i c i d a l b u f f e r meeting s o l u b i l i t y , b u f f e r pH and n o n - r e a c t i v i t y c r i t e r i a ; ( c ) u s i n g h i g h p u r i t y , s t e r i l e water f o r s o l u t i o n s ; (d) m i n i m i z i n g time between s a m p l e - t a k i n g and a d d i t i o n of HC1. No a p p r o p r i a t e ., b u f f e r was found and the UV lamp was l i m i t e d i n f i e l d and power, but the l a s t two methods were s u c c e s s f u l i n c o n t r o l l i n g mold. Evidence of S i d e R e a c t i o n Isoda et a l (3) mention a r i s e i n pH from 5.6 to 10 be-cause of the p r o d u c t i o n of NH^. T h i s was found i n the r e -d u c t i o n of the Na s a l t of o ^ - o x i m i n o g l u t a r i c a c i d to Na g l u t a m a t e . A r e a c t i o n which may be r e s p o n s i b l e f o r t h i s i s h y d r o l y s i s of the oxime to the k e t o g l u t a r a t e - 1 0 0 -N < K O - C - C - C H Z - < = H 2 ~ C - O A / C L + H X 0 " " * O NOH I 0 i MxO - C - C - CHX-CHX-C- ON*. + /Vtf.Otf. 91 Si '< o o o v i a a mechanism of the t y p e : +onx OH p/y ' • ° • T - i - -> - i - t - -> - c -Ai-oH -N-otf //A/of/ + HHjOH As i n the Japanese work, an i n c r e a s e d pH was observed a f t e r h y d r o g e n a t i o n . A b r i e f i n v e s t i g a t i o n was made to determine whether NH^ was p r e s e n t , and i f so the approximate amount. A q u a n t i t a t i v e t e s t f o r NH^ i s f o r m a t i o n of N H 2 H g 2 I 3 p r e c i p i t a t e from K 2HgI^ s o l u t i o n . A 0.50 M K,,HgI^ s o l u t i o n was made by adding 16.6 g KI t o a s o l u t i o n of 22.7 g H g l 2 and 12 g NaOH i n 100 ml. K 2HgI^ s o l u t i o n was added to s o l u t i o n s of g l u t a m i c a c i d and oxime and gave no p r e c i p i t a t e However m i x i n g 3 ml K,,Hg I s o l u t i o n w i t h 2 ml of the hydro-genated s o l u t i o n ( i n i t i a l l y 0.6211 M oxime) gave 0.010 g NH 2Hg 2I^ red p r e c i p i t a t e . When 2 ml of the reduced s o l u t i o n was b o i l e d b e f o r e adding K 2HgI^, no p r e c i p i t a t e r e s u l t e d . Thus the ammonia was d r i v e n o f f by b o i l i n g . S i n c e NH^ a l s o r e a c t s w i t h n i n h y d r i n , b o i l e d and un-b o i l e d samples were s u b j e c t e d to the n i n h y d r i n p r o c e d u r e . The u n b o i l e d sample had an A of <~r 10 % h i g h e r than the b o i l e d one and the weight of the K,,HgI^ ppt. a l s o gave a moles NH^/ moles g l u t a m i c a c i d r a t i o of 10 %. T h e r e f o r e - 101 -r T # » i » < f j _ n ni 11 — • \ _j_ f l ( A / O A £ ^ p,-ec/p.ta.Te a p o s s i b l e h y d r o l y s i s s i d e r e a c t i o n i s i n d i c a t e d w i t h about one t e n t h the r a t e of the p rimary r e a c t i o n . No f u r t h e r t e s t i n g f o r NH^ was done nor was any k i n e t i c data c o l l e c t e d due to the minor e x t e n t of the s i d e r e a c t i o n . To t e s t f o r the presence of k e t o g l u t a r a t e i n s o l u t i o n , the ketone c o n d e n s a t i o n w i t h 2-4 d i n i t r o p h e n y l h y d r a z i n e (dnph) was used o HN-<^ + H,0 II - C L - + A d i l u t e aqueous s o l u t i o n of 2-4 dnph was prepared and used to t e s t a s e r i e s of s o l u t i o n s . Appearance of p r e c i p i t a t e i n d i c a t e d a p o s i t i v e t e s t . The s o l u t i o n s used and r e s u l t s o b t a i n e d are l i s t e d : . . . . , P r e c i p i t a t e w i t h 2-4 dnph 1. blank 2-4 dnph 2. g l u t a m i c a c i d 3. < * - o x i m i n o g l u t a r i c a c i d + (15 min.) 4. g l u t a m i c a c i d + oxime + NaOH + (15 min.) 5. h y d r o g e n a t i o n product + ( 5 min.) 6. - k e t o g l u t a r i c a c i d + ( 1 min.) 7. - h y d r o x y g l u t a r i c a c i d Though f a s t e r p r e c i p i t a t i o n i n 5. than 4. may i n d i c a t e p r e -sence of a keto group ( l i k e 6.), the p rimary r e s u l t i s an unexpected ppt. f o r oxime t h a t s u g g ests an oxime condensa-t i o n r e a c t i o n w i t h 2-4 knph p a r a l l e l to t h a t f o r k e t o n e s : M O , >7 X / V O , Q j - 11 ;i ' n i l • - 'a - 1 0 2 -The presence of <=* - k e t o g l u t a r i c a c i d Na s a l t i m p l i e s the p r o d u c t i o n v i a hy d r o g e n a t i o n of <^C -hy d r o x y g l u t a r i c a c i d s a l t . Though i t i s expected t h a t the amount of h y d r o x y a c i d ( c f g l u t a m i c a c i d ) would be s m a l l , i t i s u s e f u l to c o n s i d e r whether the two compounds have s u f f i c i e n t l y d i f f e r e n t ORD b e h a v i o r to d i s t i n g u i s h them. In o t h e r words, the r e l a t i v e r v a l u e s at d i f f e r e n t ^ 's (as a v a i l a b l e on P. E. 141 p o l a r i m e t e r ) form a p a t t e r n which d i f f e r s from compound to compound, depending on the A t and i n t e n s i t y of the asymmetric t r a n s i t i o n . Dr. Hayward's h e l p was much a p p r e c i a t e d i n the use of the 3-20 Oasco to r e c o r d ORD s p e c t r a f o r samples of -hydroxy-g l u t a r i c a c i d and g l u t a m i c a c i d . The s p e c t r a i n the A range a v a i l a b l e on the PE 141, from 589 to 436 nm ( f i g . 34) are of s i m i l a r shape, though d i f f e r i n g i n s l o p e . Taking r ( 4 3 6 ) / r ( 5 8 9 ) as a c h a r a c t e r i s t i c of the ORD c u r v e , a v a l u e of 2.11 f o r g l u t a m i c a c i d and 2.17 f o r h y d r o x y g l u t a r i c a c i d r e s u l t -- no s i g n i f i c a n t d i f f e r e n c e . Thus ORD's of the two p o s s i b l e p r o d u c t s are too s i m i l a r t o p e r m i t them t o be d i s t i n g u i s h e d . In both cases s\ .. i s so low t h a t curve d i f f e r e n c e s i n the 400 -c r i t 600 nm r e g i o n are s m a l l . E x p e r i m e n t a l R e s u l t s The c o n d i t i o n s chosen f o r use i n these experiments are s i m i l a r t o those used i n ( 3 ) . However, w h i l e the Japanese workers o f t e n changed c o n d i t i o n s a t t e m p t i n g to o p t i m i s e o p t i c a l y i e l d , i n t h i s work one s t a n d a r d s e t of c o n d i t i o n s Figure 34 Optical Rotatory Dispersion Curves of (+)-Glutamic acid and (+)-Hydroxyglutaric acid: Degrees Rotation vs. Wavelength, 0.12 - 1 0 4 -was used. This was done so t h a t i f asymmetry was produced, the r e s u l t s of a l a r g e number of e q u i v a l e n t experiments c o u l d be graphed ( o p t i c a l y i e l d vs number of e x p e r i m e n t s ) t o , hope-f u l l y , show a symmetric Gaussian d i s t r i b u t i o n around the racemic p r o d u c t . T h i s would demonstrate spontaneous asymmetry. The s t a n d a r d c o n d i t i o n s chosen were an p r e s s u r e of 1100 p s i , 0.100 g R-Ni, 20 ml of f i l t e r e d s o l u t i o n c o n t a i n i n g 0.6211 M oxime and 0.6211 M KH 2P0^ w i t h an i n i t i a l pH of 6.0. E x c l u s i v e of a low p r e s s u r e h y d r o g e n a t i o n (70 l b ) t h a t pro-duced no measurable g l u t a m i c a c i d , t w e l v e h i g h p r e s s u r e experiments were completed. The experiment was not e x a c t l y r e p l i c a t e d : 3 d i f f e r e n t c a t a l y s t batches were t e s t e d , as were wet and dry c a t a l y s t s and v a r y i n g amounts of ( + ) - g l u t a m i c a c i d d o p i n g . A l s o some 2 p a r t e xperiments were done, w i t h f r e s h oxime s o l u t i o n added to the c a t a l y s t kept from the f i r s t p a r t . The experiments r e v e a l e d wide v a r i a t i o n s i n r a t e , but more s u r p r i s i n g was the d i v i s i o n of the r e s u l t s i n t o e x p e r i m e n t s showing a l i n e a r r a t e law and those d e m o n s t r a t i n g a u t o c a t a l y t i c b e h a v i o r . There was very l i t t l e e v i d e n c e f o r a markedly asym-m e t r i c p r o d u c t at any s t a g e through most of the r u n s . A c c o r d -i n g l y , i t i s p o s s i b l e to summarise the r e s u l t s f o r a l l the experiments i n t a b u l a r form and g i v e complete r e s u l t s f o r a s m a l l e r number w i t h o u t l o s s of g e n e r a l i t y (Table 1). From the t a b l e , experiments w i t h a p a r a b o l i c r a t e law (P) o c c u r r e d i n the e a r l y runs where low temperatures were used w i t h the f i r s t batch of c a t a l y s t . Use of the f i r s t can of TABLE I SUMMARY OF HYDROGENATION CONDITIONS AND RESULTS Run # C a t a l y s t B a t c h # Temp °C R a t e Law Wet or Dry C a t a l y s t % G l u Doped % Chem Y i e l d <§> 40 h r , ORT1CRL V/BLD Maximum P 1 2 3 ( a ) (b) 4 5 6 7 8 ( a ) (b) 9 ( a ) (b) 10 11 12 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 5 5 5 5 5 20 20 30 30 30 30 30 30 30 30 P P P L P P L L L L L P L L L D D D D D D D D D D D D D W W 0 0 0 0 0 0 0 0 1 1 5 5 5 5 5 16.5 10 7 14.5. 1. 75 0.5 11 13 . 7 16 .5 8.2 19 15.8 16 22 20 + 3- : 0 -41 +5 0 mold 0 0 0 + 10 0 0 -40 +5 0 0 0 - 106 -R-Ni was d i s c o n t i n u e d when the r a t e dropped i n runs 4 and 5. The most l i k e l y reason f o r lower r e a c t i o n r a t e i s a i r o x i d a -t i o n of the d r i e d R-Ni. Because of low r e a c t i o n r a t e i n run 4, the temperature i n run 5 was i n c r e a s e d . t o 20° from *J 5° i n run 4, but the r a t e remained low. The p a r a b o l i c r a t e law was f a v o u r e d o n l y once w i t h samples from the second batch of c a t a l y s t . A l l batches of c a t a l y s t were the same s p e c i f i c a t i o n ^ of R-Ni c a t a l y s t from Grace Co. The r a t e s , as i n d i c a t e d by the % chem y i e l d a f t e r 40 h r . , vary by a f a c t o r of two, i f runs 4 and 5 are s e t a s i d e . Rates from the second batch are s i m i l a r to those of the f i r s t , but a s l i g h t i n c r e a s e i n r a t e i s seen w i t h the use of wet c a t a l y s t (runs 11 and 1 2 ) . Three runs d i d e x h i b i t asymmetry o u t s i d e e r r o r l i m i t s : runs 3 ( a ) , 8 ( a ) , ? ( b ) . But d e s p i t e the l a r g e s i z e of some P va l u e s (40 % ) , the a c t u a l r o t a t i o n s are q u i t e s m a l l . A l s o , some of the nonzero P v a l u e s are s i n g u l a r -- o c c u r r i n g once i n an experiment w i t h o u t f u r t h e r e v i d e n c e of asymmetry i n samples taken at o t h e r t i m e s . Random v a r i a t i o n i n r v a l u e s was a f a c t o r to c o n s i d e r i n d e c i d i n g which r e s u l t s were s i g n i f i c a n t . S i g n i f i c a n t r e s u l t s show r changes (from t h a t at t = 0) which are c o n s i s t e n t w i t h the 0RD p a t t e r n over the ^ 1 s m o n i t o r e d . As mentioned p r e v i o u s l y , the r e a c t i n g s o l u t i o n developed a green c o l o r i n d i c a t i n g N i 2 + . The appearance of the c o l o r was g e n e r a l throughout the runs and t y p i c a l l y became n o t i c e -- 107 -a b l e as a f a i n t green at low c h e m i c a l y i e l d s (2 -.5%) and d a r k e n i n g to o p a c i t y by 10 %. As n i c k e l d i s s o l v e s , the s u r f a c e l a y e r of the c a t a l y s t i s s t r i p p e d . I f the r a t e of t h i s p r o c e s s i s slow compared to t h a t of the c a t a l y s i s , t h e r e might be l i t t l e e f f e c t on the r e d u c t i o n . The Japanese workers however made no r e p o r t of d i s s o l v e d n i c k e l , and, by measuring take-up, r e p o r t e d c o m p l e t i o n of r e d u c t i o n i n seven hours or l e s s . L i k e l y the primary reason f o r the d i f f e r e n c e i n r a t e s i s the a c t i v i t y of the c a t a l y s t s : the use by the Japanese of f r e s h l y p r e p a r e d c a t a l y s t c f the use i n t h i s work of c o m m e r c i a l l y prepared c a t a l y s t of lower a c t i v i t y . D i s s o l v e d n i c k e l may r e s u l t o n l y from the r e l a -t i v e l y l o n g times i n v o l v e d and not be connected w i t h e x t e n t of r e a c t i o n , i n the absence of a NiO monolayer. There was very l i t t l e green c o l o r i n runs 4 and 5. E x a m i n a t i o n of a few runs i n d e t a i l w i l l demonstrate t y p i c a l r e a c t i o n b e h a v i o r . Runs 3 ( a ) , 3 ( b ) , 9(a) and 9(b) are p r e s e n t e d i n f u l l . Run 3 used c a t a l y s t b a t c h #1 w i t h the R-Ni r e t a i n e d a f t e r use i n 3(a) f o r c a t a l y s i s w i t h f r e s h oxime s o l u t i o n i n 3 ( b ) . Runs 9(a) and 9(b) were s i m i l a r l y a r r a n g e d , except 5 % (0.031 M) (+)-.glutami c a c i d was added to the i n i t i a l oxime s o l u t i o n of (a) and (b) to dope the c a t a l y s t i n an attempt to i n c r e a s e p r o d u c t i o n of t h a t enantiomer. E x p e r i m e n t a l data f o r run 3(a) are l i s t e d below: - 1 0 8 -Time Absorbance ( n i n h y d r i n ) % g l u t a m i c a c i d 0 0 12.25 0.315 1.0 16.25 0.377 1.35 20.75 0.546 2.3 35 0.980 4.8 40 1.380 7.05 O p t i c a l r o t a t i o n s 0 589 nm 578 436 12.25 16.25 002 .002 -.0055 001 .001 -.007 001 .001 -.011 20.75 35 40 .0065 -.003 .002 .0045 -.0045 .001 .003 -.0075 -.0005 O p t i c a l r o t a t i o n s c o r r e c t e d f o r p o l a r i m e t e r and s t a r t i n g s o l u t i o n : 589 0 0 -.008 .004 -.005 0 578 0 0 -.008 .003 -.005 0 436 0 0 -.012 .003 -.008 -.001 P, the % o p t i c a l y i e l d i s found by P = r ( 1 0 0 0 ) 2 ( 1 0 0 2 ) = 6910 r/X X(.6211)147)31.7) where r i s o p t i c a l r o t a t i o n i n degrees a t 589 nm and X i s % g l u t a m i c a c i d y i e l d . time 0 12.25 16.25 20.75 35 40 P(%) 0 0+7 -41+5 +9+3 -7.2+1.5 0+1 These data are graphed i n f i g . (35) w i t h c h e m i c a l y i e l d vs time and f i g . (36) w i t h o p t i c a l y i e l d vs ti m e . Tables are i n c l u d e d f o r run 3(b) of c o r r e c t e d o p t i c a l r o t a t i o n s and % c h e m i c a l y i e l d vs t i m e , but o n l y c h e m i c a l y i e l d vs time i s graphed ( f i g . 3 7). Figure 36 Run 3(a); Optical Yield, P vs. Time 10 20 25 Time Hp. 30 35 40 45 5 0 - 112 -r o t a t i o n s time % Glu 589 578 436 P 0 .3 0 0 -.001 0 13 4.2 -.001 -,003 16 5.6 .003 .001 0 0 19.7 6.9 .001 .002 0 0 31.7 11 36 12 mold 40 14.5 growth The o p t i c a l y i e l d P was c a l c u l a t e d from r at 589 nm i f the p a t t e r n of r vs A as shown i n the 0RD was r o u g h l y demonstrated i n the r o t a t i o n r e s u l t s . T his worked out q u i t e w e l l i n run 3(a) except at 20.75 hr where r ( 5 8 9 ) was g r e a t e r than t h a t at o t h e r A 's. However i n 3(b) t h e r e i s l i t t l e e v i d e n c e of the r o t a t i o n a l c o n s i s t e n c y needed to j u s t i f y c a l c u l a t i o n of P. Run 9 t e s t e d the e f f e c t of 5 % doping w i t h ( + ) - g l u t a m i c a c i d , u s i n g the second batch of c a t a l y s t . S i g n i f i c a n t asym-metry was found i n 9 ( b ) , but the more p r e v a l e n t enantiomer was ( - ) - g l u t a m i c a c i d -- the dopant was ( + ) - g l u t a m i c a c i d , added i n an attempt to i n c r e a s e p r o d u c t i o n of t h a t enantiomer. Ghemical y i e l d and r o t a t i o n r e s u l t s f o r run 9(a) a r e : time chem y i e l d r o t a t i o n s 589 578 546 436 P 0 0 .074 .076 .088 .156 0 3.58 0.2 .073 .076 .086 . 153 0 7.33 2.5 .074 .076 .088 .157 0 9.58 4.3 .074 .077 .088 .156 0 20.92 8.6 .075 .077 .087 .155 0 26.92 11.6 •N/.075 .075 .086 .154 0 31.67 16.6 ^ .075 .077 .088 . 155 0 The graph of c h e m i c a l y i e l d vs time f o r run 9 ( a) ( f i g . i n d i c a t e s a l i n e a r r a t e law w h i l e the r o t a t i o n r e s u l t s show no o p t i c a l y i e l d . The outcome of run 9 ( b ) , though the same c a t a l y s t was i n v o l v e d , was very d i f f e r e n t . The r e s u l t s f o r run 9(b) are summarised: - 1 1 3 -Figure 38 Run 9(a): $ Chemical Yield vs. Time - 1 1 4 -time chem y i e l d r o t a t i o n s 589 578 546 436 P 0 0 .075 .080 .091 .159 0 4.6 1. 35 .067 .071 .081 . 146 -41 + 5 16 2. 77 .071 .075 .086 .152 -10 + 2 . 5 20 3.6 /v .068 .074 .084 .149 -9.6 + 1 . 5 24.5 6.2 •v.070 .075 .087 .153 -4.5+1 26 . 33 7.6 «/.072 .076 .087 .153 -2.7+1 (The ~ ' mark by some of the 589 nm ro t a t i o n s i n d i c a t e s un-u s u a l l y l a r g e f l u c t u a t i o n s i n v a l u e . ) Though the c o r r e s p o n -dence to l i n e a r or p a r a b o l i c r a t e laws i s not e x a c t l y f o l l o w e d e i t h e r i n run 9(a) or 9 ( b ) , t h e r e i s c l o s e r matching of the 9(b) r e s u l t s to a p a r a b o l i c r a t e law ( f i g . 39). P r i s e s to an e a r l y maximum of ^/ -40% ( w i t h r =.008°) and g r a d u a l l y de-cr e a s e s as a r e s u l t both of decreased r and i n c r e a s e d X ( f i g . 4 0 ) . Some assessment can be g i v e n on the b a s i s of the da t a c o l l e c t e d , b e a r i n g i n mind t h a t s m a l l r v a l u e s and the l i m i t e d number of runs w i l l o n l y a l l o w t e n t a t i v e c o n c l u s i o n s . A c c o r d i n g to H a r r i s o n ' s e x a m i n a t i o n of p o s s i b l e c a t a l y s i s k i n e t i c s , a u t o -c a t a l y t i c b e h a v i o r i s a ne c e s s a r y p r e c u r s o r to spontaneous asymmetry. E x p e r i m e n t a l l y , s i x h y d r o g e n a t i o n s out of 15 showed evidence a p p r o p r i a t e to a u t o c a t a l y t i c f u n c t i o n i . e . the p a r a -b o l i c r a t e law. The few examples of nonracemic product o c c u r r e d mostly i n runs d i s p l a y i n g the p a r a b o l i c r a t e law, though these comprised o n l y 40 % of the t o t a l . A c o n c l u s i o n r e l a t i n g l a r g e P v a l u e s t o p a r a b o l i c i t y would be tenuous because of i n s u f f i -c i e n t number of e x p e r i m e n t s . I t may be s i g n i f i c a n t t h a t the only run p r o d u c i n g nonzero P w i t h a l i n e a r r a t e law does so on l y at one time w i t h r = 0.003 which i s n e a r l y minimal to e s t a b l i s h a s i g n i f i c a n t change i n r o t a t i o n . - 115 -Figure 39 Run 9(b): # Chemical Yield vs. Time - 1 1 6 -Figure 40 Run 9(b): % Optical Yield, P vs. Time +10 0©--10 p I -20 -30 -40 -50 1 J L J L 1 0 Time 1 5 Hr. 2 0 25 - 117 -A b a s i c p a r t of H a r r i s o n ' s k i n e t i c approach i s a m p l i f i -c a t i o n of an i n i t i a l e n a n t i o m e r i c i m b a l a n c e , l e a d i n g to spontaneous r e s o l u t i o n . The one case, 9 ( b ) , t h a t best demonstrates spontaneous asymmetry under the i n i t i a l e n a n t i o -meric imbalance of pure ( + ) - g l u t a m i c a c i d does no t , however, g i v e i n c r e a s e d r o t a t i o n s but decreased r o t a t i o n s . T his suggests 3 p o s s i b i l i t i e s : ( 1 ) t h a t the ( + ) - g l u t a m i c a c i d a m p l i f i e d p r o d u c t i o n of the o t h e r enantiomer or (Z) t h a t some ot h e r c h i r a l agent was r e s p o n s i b l e ( e . g . minute contaminant) or (3) t h a t c h i r a l s u b s t a n c e s do not a f f e c t p r o d u c t e n a n t i o -meric d i s t r i b u t i o n . The second p o s s i b i l i t y seems l e a s t l i k e l y . In e v a l u a t i n g the h y d r o g e n a t i o n r e s u l t s i n terms of the one and two product molecule s i t e m o d i f i c a t i o n d i s c u s s e d i n the I n t r o d u c t i o n , the most i m p o r t a n t f a c t o r i n d i f f e r e n t i a t i n g between the two models i s the O p t i c a l Y i e l d vs time data and not the Chemical Y i e l d vs time d a t a . The one product molecule m o d i f i c a t i o n , which produces enantiomers i n the o r i g i n a l D/L r a t i o , and the two product molecule m o d i f i c a t i o n , which a m p l i -f i e s the o r i g i n a l D/L r a t i o , both assume complete s t e r e o s p e c i f i -c i t y . The n i l O p t i c a l Y i e l d i n most of the doped ru n s , and " n e g a t i v e feedback" i n the 5 % doped run of g r e a t e s t O p t i c a l Y i e l d s u g g e s t s , i n the case of a u t o c a t a l y s i s , low s t e r e o s p e c i f i -c i t y . The l i n e a r ( z e r o - o r d e r ) p l o t s found i n most of the runs w i t h the second batch of c a t a l y s t c o r r e s p o n d to the n o r m a l l y expected b e h a v i o r of a heterogeneous c a t a l y s i s i n which the e n t i r e s u r f a c e i s a c t i v e t hroughout a l l r u n s , so t h a t the r e -- 1 1 8 -a c t i o n r a t e i s independent of changes i n the s o l u t i o n . For an a u t o c a t a l y s i s , t h i s i m p l i e s s t r o n g a d s o r p t i o n of the p r o d u c t . The a d s o r p t i o n r e s u l t s g i v e n i n s e c t i o n HI show t h a t , even i n the presence of c o m p e t i t i o n from phosphate and oxime, Glu i s adsorbed to the e x t e n t of a monolayer when i t s c o n c e n t r a t i o n i s g r e a t e r than t h a t c o r r e s p o n d i n g to 3 % r e a c t i o n i n a h y d r o g e n a t i o n r u n . The p a r a b o l i c r e a c t i o n c u r v e s found i n the f i r s t sample of c a t a l y s t cannot be g i v e n a unique e x p l a n a t i o n on the b a s i s of the p r e s e n t data a l o n e . A t law i n d i c a t e s t h a t the r a t e of r e a c t i o n i s p r o p o r t i o n a l to t ; f o r an a u t o c a t a l y s i s , t h i s would imply t h a t the c a t a l y t i c a l l y a c t i v e p a r t of the product i s b e i n g formed i n a z e r o - o r d e r f a s h i o n . This c o u l d be r e l a t e d to the appearance of N i 2 + i n s o l u t i o n . I f the R-Ni i s d i s s o l v i n g at a c o n s t a n t r a t e and the a c t i v e c a t a l y s t i s a N i 2 + - glutamate • complex i n s o l u t i o n , then the p a r a b o l i c law i s r e a d i l y e x p l a i n e d . F u r t h e r work on homogeneous c a t a l y s i s of t h i s k i n d might be u s e f u l . - 119 -BIBLIOGRAPHY 1. K. L. Kovacs and A. S. Garay. 1975 Nature. 254. 538 2. R. E. Pincock, R. R. Perkins, A. S. Ma and K. R. Wilson. 1971 Science. N. Y.. 174. 1018 3. T. Isoda, A. Ichikawa, and T. Shimamoto. 1958 Rikagaku Kenkvusho  Hokoku. 2i, 134 4. P. C. Prank. 1953 Biochim. Biophys. Acta. 11, 459 5. P. P. Seelig. 1971 J. Theor. Biol.. 2±, 197 6. P. Decker. 1973 Nature New Bio l . . 241. 72 7. A. R. Hochstim. 1974 J u l . Conf. 13 (ed. W. Thiemann) Julich: Kernforschungsanlage, 289 8. L. G. Harrison. 1974 J. Moi. Evol.. ±, 99 9. A. T. Austin and J . Howard. 1961 J. Chem. Soc. 3278-84 10. L. A. Mokhov. 1958 Lab. Delo. £, No. 6, 46-7 11. P. H. Kasten. 1961 Intern. Rev. Cytol.. 10, 1-100 12. R. L. Shriner, R. C. Puson and D. Y. Curtin. 1961 The Systematic  Identification of Organic Compounds: A Laboratory Manual. 4th ed. 13. R. M. Matulis and J. C. Guyon. 1964 Anal. Chem.. 36. 118 14. J . G. Wood, M. R. Hone, M. E. Mattner and C. P. Symons. 1948 Australian Journal of Sci. Research. B, 1, 38 15. V. E. Price, J . B. Gilbert and J . P. Greenstein. 1949 J. Bio. Chem.. 179. 1169 16. M. I. Barabanov and I. M. Litvak. 1962 Tr. Kievsk. Tekhnol.  Inst. Pishchevoi Prom.. No. 25, 17-19 17. M. S. Dunn, E. H. Prieden, M. P. Stoddard and H. V. Brown. 1942 J. B i o l . Chem.. 144. 487 18. W. T r o l l and R. K. Carman. 1953 J. B i o l . Chem.. 200. 803 19. J . Preel, W. J . M. Pieters and R. B. Anderson. 1969 J. of Catalysis. ]&, 247 20. Y. Izumi, M. Imaida, H. Pukawa and S. Akabori. 1963 Bull. Chem.  Soc. Jan.. 26, ( l ) , 21 21. P. R. Norton and R. L. Tapping. 1975 Farad. Disc, of the Chem. Soc. No. 60, p. 71 - 120 -APPENDIX To Isoda, Ao Ichikawa and T. Shimamoto, Rikagaku Kenkyusho Hokoku ( j . Inst. Phys. and Chem, Research, Tokyo), 54, 134 (l95B). Translated by Y. Koga; translation edited and typed by L. G. Harrison (Department of bhemistry, University of B r i t i s h Columbia, Vancouver 8, B.C., Canada). STUDIES ON ASYMMETRIC SYNTHESIS. PART 1. REACTION CONDITIONS FOR ASYMMETRIC REDUCTION OP a-KETOGLUTARIC ACID. (Abstract, not translated) INTRODUCTION Asymmetric syntheses have so f a r been effected by physical, chemical and b i o l o g i c a l methods. In the chemical method, the properties of diastereoisomers are u t i l i z e d . The compound goes through an intermediate diastereoisomer, or an o p t i c a l l y active compound i s used, under the influence of which the reaction proceeds. The main factor determining the s e l e c t i v i t y of asymmetric synthesis (P$) i s of course the o p t i c a l l y active material added, but the reaction conditions have also to be considered. In many cases where the same o p t i c a l l y active substance i s used, a s l i g h t variation i n reaction conditions, such as temperature, pressure, etc., causes a change i n P; and for some cases this change i s even more sign i f i c a n t than that attained by switching the o p t i c a l l y active substance. Therefore studies were planned on the influence of reaction condition alone on asymmetric synthesis. In the f i r s t series of experiments, the diethyl ester, Na s a l t and oxime of a-ketoglutaric acid were reduced under various conditions: the catalyst, i t s quantity, reduction conditions [ i . e. hydrogen pressure and temperature, as indicated i n headings of tables - L. G. H.], solvent, concentrations of reactants, and pH of the reaction solutions were varied and the o p t i c a l rotation of the products (oc-hydroxyacid derivatives and glutamic acid) was measured, whence the P's of the reductions were calculated. The results showed some success i n asymmetric synthesis (P: maximum 10$), Thus i t was shown that under some conditions asymmetric synthesis i s possible without adding an o p t i c a l l y active substance. In the second series, dl-a-hydroxyglutaric acid ethyl ester was equilibrated with the ketoacid i n the presence of hydrogen and Raney-Ni at atmospheric pressure and high temperature, [A free translation, corresponding to the more e x p l i c i t statement i n the results s e c t i ] Generally P's are higher than i n the f i r s t s e ries. In the t h i r d s e r i e s , o p t i c a l l y active substances (D-camphor, D-borneol, L-borneol) were added to the same reactant as i n the f i r s t series, and under the same conditions. In some cases P's are reduced and i n others increased. [Note on chemical names printed i n the Roman alphabet i n the Japanese text: Some of these names use English forms (e. g. glutaric acid) while others use German forms (hexan, benzol). Also the archaic form oxy- i s used for modern English hydroxy-. Yo K., 1J. G. H.J I SERIES I Diethyl ester, Na salt and oxime of a-ketoglutaric acid reduction Diethyl ester, Na salt of oc-hydroxyglutaric acid and glutamic acid ( l ) Effect of solvents a-ketoglutaric acid diethyl ester was dissolved i n various solvents at the concentration of 2C$, and the solution was placed i n an autoclave of 150 ml capacity. In the presence of Raney-Ni (w^), the solution was reduced by gas i n the autoclave. The autoclave was shaken at 50 cycles per minute. After the reaction, the solution was d i s t i l l e d and [ a ] D of the b. p. 105-107° portion of the d i s t i l l a t e was determined and P was calculated using the value = +4.6 for the pure d- or 1-a-hydroxyglutaric acid. Results are i n tables I - VII. Generally, D(+) product was obtained i n non-polar solvents, hexane and benzene, while L(-) was predominant i n polar solvents. - The rate of uptake of i s an important factor to determine P: uptake of the theoretical equivalent amount of H 2 (to 10 g of reactant) i n 4.5-7 hr seems to be an optimum rate, and at a higher rate racemic product was obtained. [Comment: It i s not clear what this means. Rate of uptake cannot be varied independently of pressure and temperature, and this statement may 'represent a concealed effect of pressure or temperature dependence - Y. K.f L. G. H.] The amount of catalyst i s also important i n that less than of catalyst (in relation to reactant) brings about racemic product. Translation of headings for tables I - IX:-Reactant Catalyst Reduction conditions Solvent (g) ($ of reactant) I n i t i a l press. H 2 (atm) TUC (ml) uptake time (hr) Product a-hydroxyacid ester  Reduction of a-ketoglutaric acid i n : (a) table I hexane (b) table II benzene (c) table I I I ethanol (d) table IV ethyl acetate (e) table V ether (f) table VI methanol (e) table VII no solvent - 122 --3-(2) The same experiment as in ( l ) except that "stabilized Hi"(NiO) and Ni formate were used instead of Raney-Ni. The asymmetric properties of the oc-hydroxyacid produced were essentially the same with NiO as the catalyst as i n the previous experiments with Raney-Ni. When Ni formate was used as catalyst, however, with ethyl acetate as solvent D(+) product predominated at low pressure and L(-) at high pressure; i n hexane also, L(-) was obtained at high pressure. (Headings for tables VIII and IX are the same as for tables I - VIl) (3) Effect of nH The Na salt of a-ketoglutaric acid was reduced i n 10$ aq. soln. with varying pH, using Raney-Ni and U-Ni-A. At low pH values, Raney-Ni (w,.) tends to lose i t s catalytic a c t i v i t y , hence U-Ni-A was used at pH 3.5-6.5. Table X. Reduction of Na salt of a-ketoglutaric acid i n HgO. Reactant (e) Catalyst ($ of reactant) H_0 (ml) pH Reduction conditions Product a-hydroxyacid Na salt Inito press. H 2 (atm.) TUC p$ (4) The same as (3) except that the concentration of aq. soln. of the reactant salt i s raised to 25$ and the pH range studied was 6-8 and thus only Raney-Ni (w,-) was used. Table XI 0 Reduction of a-ketoglutaric acid Na salt i n a highly concentrated aq. soln. at pH 6-80 (Same headings as table X.) Table XII. Reduction of a-ketoglutaric acid Na salt i n a highly concentrated aq. soln. at pH 8 0 [items 13 and 14 i n table refer to a mixed H^ O/EtOH solvent, as allowed for i n the table headings - L. G. H.] Reactant (g) Catalyst ($ of reactant) Na bicarbonate Solvent .Reduction con ditions .. Prodi <?t amount added (g) pH H20 (ml) EtOH (ml) I n i t . nress. H 2 Utm) [«3D (5) a-oximinoglutaric acid diethyl ester was reduced i n a few kinds of solvents by Raney-Ni (w^). In non-polar solvents, the racemic product was obtained, but i n ethyl acetate I K - ) type resultedo Table XIII. Reduction of a-oximinoglutaric acid diethyl ester. Reactant (g) Catalyst Solvent (ml) . Reduction conditions Product a-liit.win nci.d I n i t . nrcsso H 2 (atm.) T"C [a] DHCl P$ - 123 --4-(6) The Na salt of a-oximinoglutaric acid was reduced in 10$ aq. soln. and D(-) glutamic acid was obtained. I n i t i a l l y , the pH of the soln, was 5.6, but at the end of the reduction i t became about 10 because one of the products i s NH^ « This could be the reason why only D(-) product was obtained,, Table XIV0 Reduction of Na salt of a-oximinoglutaric acid. (Headings as table XIII, but with an extra column for pH; and i n the column headings, the solvent i s specified as H^ O and the catalyst as Raney-Ni.) (7) The Na salt of a-ketoglut aric acid was reduced i n NH^  solution and L(+) glutamic acid was obtained. Table XV. Reduction of Na salt of a-ketoglutaric acid Na salt i n NH^  soln. (Headings as for table XIII, except that they now specify the catalyst as Raney-Ni and the solvent as 28$ NH.OH.) 4 ••} SERIES II The pos s i b i l i t y of asymmetric transposition of dl-a-hydroxyglutaric acid diethyl ester was studied by u t i l i z i n g the dehydrogenation-hydrogenation equilibrium [between hydroxyacid and ketoacid - Y. K., L. G. H.] without the influence of an optically active substance. dl-a-hydroxyglutaric acid diethyl ester was treated i n at atmospheric pressure and high temperature i n the presence of Raney-Ni (vO. In nolar solvents the ester 5 ' was converted to L(-) type and in non-polar solvents to D(+) type. This trend i s the same as in series I experiments, but P's are higher than i n series I. A part of the ester after the treatment was i n the dehydrogenated state (a-ketoglutaric acid ester), and the amount of this was determined by using 2,4-dinitrophenylhydrazine. +, ++, -t-H-in table XVI signifies the amount of ketoacid i n increasing order and - implies that no ketoacid was found. In cyclohexane, the hydrogenation-dehydrogenation reaction occurred but the ester stayed racemic 0 Table XVI. Asymmetric transposition of dl-a-hydroxyglutaric acid diethyl ester Reactant (g) Catalyst ($) Reaction conditions Solvent I n i t . press. TUC time (ml) H2 (hr) atmospheric Product a-hydroxyglutaric acid diethyl ester Reactn. with P$ 2,4-DNP' SERIES III ( l ) The same experiments as series I ( l ) , except that optically active substances such as d-camphor, d-borneol, i-borneol were added. Table XVII„ Reduction of a-ketoglutaric acid diethyl ester i n the presence of optically active substances. - 124 --5-Reactant (g) Catalyst •ney-Ran Ni (Wr) Additive (g) Solvent Reduction conditions Product (ml) Water (ml) I n i t . ureas. H 2 (atm) P$ (2) The same experiments as i n series l(5) and (6), except that d-camphor, d-bornebl and i-borneol are added. Table XVIII. Reduction of diethyl ester or Na salt of cx-oximinoglutaric acid i n the presence of optically active substances. (Headings as table XVII except that third and fourth columns are respectively solvent (ml) and additives (g); and the product i s of course glutamic acid salt or ester.) NOTE BY L. G. HARRISON After the Japanese text, I append three distribution diagrams for the results. These histograms are not quite symmetric, as they should be i f one i s to prove spontaneity i n the occurrence of asymmetry in any individual experiment. The average asymmetry of a l l experiments (143 experiments) i s displaced from zero by an amount exceeding the standard deviation: -1.20$ +_ 0.4$. Results from tables X, XI and XII only give -0.98$ + 0.7$. For the 18 experiments numbered 20 - 37 i n table X, however, i n which a-ketoglutaric acid Na salt was reduced i n aqueous solution at pH 8, the centre of the distribution curve i s at -0.94$ + 1.7$o It s t i l l shows a bias towards negative rotation, but the bias i s now within limits of error. This distribution curve i s interesting; i t i s multiple-peaked, looking something l i k e the two-coin-flip distribution. A pity there weren't ten times as many repeats of this experiment. - 125 -(32) a - K e t o ' G l u t a r i c A c i d S S ^ f + i tO^J ' F i S ^ i : ^ © ^ S g r i ' - j ^ f N c - J ^ - c acid aW5r=FiSf't'.G-»B2'2t L C v ^ v i e ^ j f t - - t ? J S 5 D Ufc i c: P?* IJt'h S «,•»:, «fj 3 Mi »CV>-t? dl-a-oiy-glutaric acid diethyl ester Sr, lull: < rJt^f i5tt^K>85 'KLtd, a-OH ?,« • t S t l o - e S S , ft&K a-keto glutaric acid diethyl ester &:ft$tfffe4%c>;)t-flrrKi:93C L. '.ts m S ^ ' I f f t W j ^ f t V ^ t ^ m W i C t t diastercoisomer tOttKWfflJffi l ' lt ' l l L t ' i W J diastcreo-•fc-t; 3$-©«-*%• »stftiir^ffKctec^:+ *.>&ficM):*ft-earn*ic4 sr<h«« u». wti- L--UVI«IW; a-keto glutaric acid © diethyl ester, Na salt, oxim Srffl^rCJ-Sfftrjg/cL, ftW.©f'|i.Wi, J.!;, jH AJ&lt, ft'f (KOHtffl,iaa^«iftl«, R{Ei«<0 pH IJECOWT-ICJ: •)'.!•:»*LA: «-oxy acid tfiflW, glutamic acid S S & W f c L T , a-keto glutaric acid &jg/Ui©ti, 7 2 / (St <> Affe»:*> •) ' f e t t ^ I ^ S fe * t t - « J . 5 C 4 t , ««<t-&tl'!!*)?>*>f>ray* Lfc a-oxy acid ester, Na salt tmiKit^t^fil^hM cis-trans isomer kHfcOSXO mirror isomer  :h I i t A, t'SS 'S J •& t ^'x tz!it/>X\h%e lfcV»t!3f5r3l»t L T dl-a-oxy glutaric acid diethyl ester fcWSL. ^ il rV/r;;a, K^/v! ilFiC Raney-- 126 '-(33) >f;ft-o&s n ra+ a wet Ois--«i) 135 M f £ o ;T5 •''A:Sit: C .t ; l - -r jt'7;<7:l'l-.f,<j f t J : L '.: D-camphor. O horneol, L-borneoI tKW *<OMfTlZ «-k c t " * , u , a r i c a t i d d i t , M c s t e r S - ' « » r w * f t J r « H | » < u L. xwmtioisvn mtz£ 9 M f e * n It I. fi-keto Rlularic acid <r> diethyl ester. Na salt, oxim • £•ft W;, FitW;, St Jii-'fctT•£>SMfcilM-TJ3JC, a-oxy Rlutaric acid CO ester, Na salt U.ltf Rlutamic acid fc-'fclifc U KARCOiEA. P# fr^t^-^JSJCro^BI ft 7; ft-<r ft i l l U : o ( 1 ) a-Keto Rlutaric acid diethyl ester £• -£«I<E 20;* ftffi , ¥ W 7 Raney-Ni (W,) I; J: O&yG&ft SrJgfl : U - C * ^ ; « U l-fc L ' C S B i : t * K T ? * r . » J « ! f LA:. ttXS/ftliftM'K 30 [fi|/#, FSft ISOe.c. © x ---O'Mmii'Wkftfi-f^ l.HtW&tiiW. hcxan. benzol ')> "i DC+) 'V. a-oxy acid ester flS©«tt»«t t ' ' ( t IX-) -ilVZ'MUu 4/>ft!*x + * (AcoEO, ether ' ( ^ ( i , fl/KKfcV'T D ( + ) S». Ufc f t f j l t i . ' . t f L J ?»M K * u a t A * t M « LW <. J-vaJi'rc P * ttflv. - K i r U - ) » © X A M W t D ( + ) S? S£ Jo S: jj(f ( a ) Hexan frrWt-i^t'G «-kcto glutaric acid diethyl ester OO$JVLI i ( R ) 10 (;») 5 8 5 5 (atm.) 22 12 40 40 ill K TO 85 26 70 70 Iff 7K3E54JR ' ' - '^ a-oxy acid ester (ml.). ( I ' M ) C«]D 50 4.5 +0.23 5 n 7.5 + 0.13 2.8 it 5.S // 2.8 II 4.5 +0.1 2.1 Ht^nm^ei< i\ p?< ( b ) Benzol ti'MiZ); p. 105°~107*C » S I » ! - . o i . r [ „ i „ 5 r^ . i ' L ^„ Raney-Ni ?i l i t t ia^cfeKtr ( i a oxy jjlutaric acid diethyl ester Wjt/'/tfjICttiViCO Ca]D±4.6 i LTIh?^ L f & a keto glutaric acid diethyl ester <Di$ji id ii. n (K) (?») (atm.) * It-,U IE C O i 10 3.5 21 40 2 II 5.7 22 65 3 II 7 22 30 4 II 10 17 25 tSE 7jt5^-!5jf^-'k'S a-oxy acid ester (ml.) wnn) Ca3n 50 7 + 0.15 3.3 II 7.5 + 0.05 1.08 II 7 II 1.08 it 5.5 + 0.35 ' 7.6 ( c ) EtOH a-kcto (jlutaric acid diethyl ester i/)$tit ff5 111 n - 127 -136 (34) (#H-MW m&Tcw, to « ( g ) 10 oo 3 5 5 5 7.5 7.5 10 10 • ' K ^ ^ . ' . ' . ' . i -(atm.) 20 12 22 12 10 22 35 * ft-ai BE CO . 100 65 70 60 55 60 (ml.) 50. 7 k 5 f - ; » W ( n j l i i l ) 5 6 8 i 6 'k/J; tt-oxv acid ester CoOn 0 -0.1 -0.15 -0.05 it II -0.35 -0.15 I ' CO 0 2.2 3.2 1.1 II n 7.6 3.3 ( d ) AcoEt JSWI i fc l+ i a-keto glutaric acid diethyl ester ^Sfyu as iv m. | Cg) to tt • oo Catm.)_ fJk ft-! ai re CO. ... (ml.) Cum) ' Ullik rt-oxy Ca)n . acid ester P O O 1 10 3 12 85 50 7 j 0 i 0 2 II II 44 : n // 6 0 ' 0 3 II 5 5 ' a II // -0.15 3.3 4 II '' 22 75 II i it -0.1 2.2 5 n n 41 // II 4 + 0.15 3.3 6 n 7 35 65 • II // +0.1 | 2.2 7 II 10 8 85 II 5 " II 8 n n 12 65 II 7 -0.2 ; 4.3 9 n a 20 55 II 6 -0.3 ' 6.5 10 II " 35 II It 7 | -0.15 ' 3.3 .) Ether t&W-AZ&tr} Zt a-keto glutaric acid diethyl ester COiStvU ip v 3i ( g ) to «t-oo i;3 vt, _.Q>im.:)._ * ft-1 ai re CO.... i'r'f tt (ml.) CSI'i!) rt-oxy acid ester tajD I 'OO 1 10 s 20 ! 95 50 4 -0.15 3.3 2 a 7 34 ! 65 4 + 0.1 2.2 3 n 10 6 70 II 5.5 -0.25 5.4 4 a " 10 67 it „ tt tt f ) -keto glutaric acid diethyl ester cOj^Jii AS VI 3 i Cg) to tt Catm.) * fr-ill re CO Cml.) 0'iflii]) a-oxy acid ester P O O ' 10 10 ... ) 0 30 45 50 2.5 -0.05 1.1 Cg) MfeW'tetfi a-keto glutaric acid diethyl ester CD'SJC - 128 tii-V) (35) 137 » VD C g ) tt. »s oo •ik-JUPJI.E (atm.) •% fr-ill ffi C O 1 10 5 37 90 2 n 10 12 60~70 3 n 10 60 90 vkSSS'K a-oxy acid ester O'JIiiJ) Ca3n • P C ?0 5 7 4.5 0 ! -0.45 I -0.42 i 0 9.8 9.2 ( 2 ) 7iMi\t N i (N iO) , #'f» Ni (F-Ni) >, (1) [;.]«• a-keto glutaric acid diethyl ester £ 2 - 3 ©««.<|'-riaxL, )SM*OM WCXZi&Kt;BW+ajS? Lfc. ««.»!>Kttii:'M a-oxy acid » « K » | I 8 H W Raney-Ni fcfflW.:. CD <0«J&tCrt-J, NiO TrUI^-HS iNj-TT&S^F-Ni - c u AcoEt tiM'VT:im]\ :.X DC+) « , A'/HT; L ( - ) 5'!5r* U &©« rn ] £ * hexan ' l 't?liil^T..LC-) WO oxy acid fr<kl* ( a ) NiO II.t& a-keto glutaric diethyl ester OJSLJC tfS Vffl X ! Cg) ; tt O O Camt.) iv. fr-i l l IE .C°c) ( m l / | (nifrifl) ikiiS a-ox Ca]D l acid ester P O O 1 ; io i 3 5 130~140 HexSn50 " ' s . s ~ " 0 0 2 it • 3 22 II 'I 3 0 0 3 tt 5 5 " 5 +0.05 i i 4 II ! 22 II II 3 II / / 5 II 23 II II 2.5 +0.25 5.4 6 ! // 1 10 5 II II 3.5 -0.2 4.3 7 it 32. it II 1 + 0.1 2.2 8 i " it 22 • — Benzol 50 McOHSO — -0.25 5.4 9 // II 32 140~150 2.5 -0.1 2.2 10 // 5 10 II ^tcoSt sb. 6 II it 11 ! " 10 10 II \ 6 . S II II 12 // II 32 140 it \ N-3 II II ( b ) F -N i A Z X i , a-kcto glutaric acid diethyl ester CDMit id ix m. >«.:•'vrttlt (g) oo i3 5t 7k * w n i (atm.) -x ft-ai BE C O m .«! (ml.) i 1 10 10 10 120~130 EtOH 50 j 2 2 '' a 32 • II II 1.5 3 // 30 it AcoEt 50 I 5 4 • it 10 II II 6 5 // tt 32 II it 5 6 tt 10 it  - Hexan50i 3 7 it 10 115—125 " ! 5 8 tt 32 it it ; 2. ikfiS a-oxy acid ester C O ] D i P O O -0.2 -0.08 +0.15 +0.05 -0.3 +0.05 + 0.35 -0.2 4.3 1.7 3.3 1.1 6.5 1.1 7.6 4.3 ( 3 ) a-Keto glutaric acid Na salt Jr U-Ni-A ir>£V>|J Reney-Ni (W s ) HJ :U f<J 10?« 7}cfi?JB:r(J't: - 129 -138 amfssfl, ffi/M an, tvo-ft® (36) (t ft - . - i -wt Hl/rO pH K M -rff lTt U '.fcl# ar-oxy Rlutaric acid N'a salt 1' ?» tV**W U : . pH «OtU-£ » i Raney-Ni « i S t t < 0 « T fcJR* A:#>. pH 3.5~6.5 iU-n>Xlt U-Ni-A f«fHI I/:. H i O »«8»c4«ltS « keto glutaric acid Na ffi^iSvi; Cg) oo . * (ml.) pH -*vK>JJ'!: (atm.) & iT m m C O -'l-Jiic tt-oxy CoOn •; acid Na #1 P O O 1 6 U-Ni A 15 50 3.5 64 80 + 0.6 7 2 I) // - // 4.5 10 it + 0.3 | 3.5 3 n II tl " 49 75 0 j 0 4 II II it n 12 // o ! 0 5 II II if it 40 tt +0.1 : 1.1 6 II II it it 38 tt +0.5 ; 6 7 n it tl II 10 • 85 +0.05 ; 0.6 8 II it ll II 35 75 // : it 9 II II it 5.6 28 55 // it 10 n II ll n 10 „ + 0.4 4.7 11 it II ll 6.5 II 35 +0.6 7 12 II it it it 27 40 +0:1 | 1.1 13 II R-Ni W, 10 ll 7 10 tt i -o.o5 ; 0.6 14 II / / ll it 20 ft -0.14 I 1.6 15 II II it tt 60 it + 0.28 | 3.4 16 II 5 ll ,, . it tt +0.21 ; 2.4 17 II it ll II 10 45 o ; 0 18 II 10 it II 30 40 ' 0 i 0 19 II tt ll " 70 tl . +0.05 ; 0.6 20 n n it 10 ll o ! 0 21 II n n It 30 45 -0.83 : 9.5 22 II 5 tl " . 10 40 0 1 0 23 II it ll 30 tl -0.75 8.7 24 II 10 " 10 it -0.83 9.5 25 it tt „ - 30 45 -0.21 ! 2.4 26 II II II " 20 40 -0.83 9.5 27 it 5 II II it if -0.21 j 2.4 28 II it "' " 60 ,, -0.83 9.5 29 II 3 it it 10 0 0 30 II II it it 60 . -0.41 4.7 31 it 10 it II it + 0.28 3.2 32 II n 10 it tl 30 + 0.6 7 33 II n tl it tl it + 1.0 11 34 II II n II n 39 // it 35 it it it II 10 40 + 0.6 7 36 II II n it it 31 0 - 0 37 II II tl n it 35 + 0.2 2.2 - 130 -ifs^-y-) (37) 139 pH 3.5~6.5 T7lt D ( + ) 'V!, 7~8 T;l.t L ( - ) pH 10 "C l i D ( + ) «!<D a-oxy acid Lfc. pH 7~8 K I ; DC+) mi-:kfiKt*Mi»jrtU>2>. pj.it-Kii+ttS^itrA-^'Jinj-e, *fc ftUHICM'-CA-i* < I ' i f l i t t t .£<•••. ( 4 ) MTJIWfcft-SU a-keto glutaric acid Na JJJ© Wfi *tt'fflc £3176 Uiifi, ^ISIttJ: 9ESVN8(X25# M - k K i i K Raney-Ni (W s ) £ J I K - t B )C , •AMlWrttiL-WM', pH 6~8 - C l i - ^ S r K . ? , - f ^ T L ( - ) <?! a-oxy acid Uu l i ff lff i «Ki«IS.Vjnj;-r!;Jt'<-C- -SJi lKV-J; 5»:«fcn«. pH 8 tJUWJfwaj-^ P f * * S « W S 30atm. -Stt^jTr'S ^IIW 9 Wiv £ fc/k-T"A*? —/Liii{-fHliVM'tz}ii<''CJ>&]i Lfc7)>, MHEtc L ( - ) <$<D oxy acid $ r M L / : . ' XI ii. i-itstK'ktffte (p l l 6~7) IC-frltS a-kcto glutaric acid Na fflK>J35c P H * M 5C & fr- ifeJiS oxy acid Na • Cg) O O , 7 * (ml.) (atm.) ill w ca Ca)D P O O 1 5 ' 5 6 6 90 +0.4 4.7 2 " ! II 20 // 10 40 -0.55 6.6 3 // II tt II 30 40 -0.35 4.2 4 it tt 6.6 : 30 35 -0.8 9.4 5 // n tt . // 10 35 -0.1 1.1 6 // • tt 10 it 10 35 -0.48 5.6 7 '/ i it . 5 II ; . 30 125 -0.1 1.1 8 it it /.' tl i 1 0 155" -0.4 4.7 9 n 10 10 1 | 80 30 -1.7 19.7 10 it 1 // II 80 30 0 0 11 7 j 11 15 it 80 30 -0.6 7 12 It 20 ft . 15 30 - ? — 13 // \ 2.5 II II 80 30 — ? ' — 14 It 5 \ " it I 80 40 -0.25 2.9 15 It it II ti 15 40 -0.25 2.9 16 8 2.5 it tt | 15 • 35 - ? 17 5 1 12 it 15 35 0 0 #S HI Hi y. <9«^r€^ 'AflJBEvkir'fvffi CpH 8) IC^-Iti a-kctcr'Klutaric acid Na fflmSvt Pi'&jt IS & Cg) 1 5 2 // 3 II 4 II 5 n 6 II 7 II 8 II 9 II l-ion i7k*-*ir£, at BE (ml.! ! Catm.) i (°C) in Tc % fr- 4-:n£ oxy COOD 40 40 40 30 55 65 40 40 150 -0.3 0 0 0 -0.48 —0.2 0 - 0 . 4 0 acid Na iU. P C * ) 3.5 0 0 0 5.6 2.2 - 0 4.5 0 - 131 -140 (38) (SpS-t-PHtt «• St .. (6.X c?o 8S4nSt . . ( g ) i •/ - V pH iff * (ml.) « EtOH (ml.) (atm.) •ft ft- 1 s i m . C O . 'l:li£ oxy Ca)i. acid Xa i£ I'CfO 10 5 5 0.2 | 8 5 — 10 • 140 : -0.2 2.2 11 it II " n 20 • 10 40 '• 0 0 12 it \ It it 20 — 30 40 ' 0 ; 0 13 II II — 7 6 20 60 50 -0.33 • 3.8 14 it II — it 6 20 10 50 | -0.35 4.1 ( 5 ) a-Oximino glutaric acid diethyl ester £• 2 • 3 ©fi¥tt>|i.T* Rancy-Ni (\V») ( ; j ; S j£ I, glutamic acid O i f S# f lH£ I»#coV>T I&t t LA:.. MMktiM'PXIt, OHO}? * HtkM, -)i AcoEt <\<XU D ( - ) .!('!« glutamic acid fc'Ao&Ltz. •ji K ?A XJII # a-Oximino glutaric acid diethyl ester Sj'R'jt ( g ) (?« fl? tt (ml.) fit1 vu 7h?R*iin (atm.) •Vfr fl-41 K C c ) .'I-:)L'< Glutamic acid C«JI>IICI 1 5 10 AcoEt 50 14 120 -0.5 1.7 2 // it // 30 " 120 -0.4 1.3 3 it it Hexan 50 25 120 + 0.05 0.2 (6) a-Oximino glutaric acid Na JJJfr 10?i /k(>'f((i:'ll":avL: L. D ( - ) W> glutamic acid 5.-j'.//^ „ P H 5.6. 7 i r o v > r ^ L A : « : ; a J u r r m t c i i . K NH, w f c w P n 10 1 ^ : 4 1 1 , ziifr D(-) W ) LA:-W/).i>Mit£*.\ flS XIV ii ff-Oximino glutaric acid Na tilfsjv.X: \ f ! K a n c y - N i ...„(.?/) 7k ( m l . ) pH 1 i:3 -A: ', (a tm.) ft-iU BE C O .'kliU G l u t a m i c acid Caj i iHCl P(?<0 1 ; 5 5 50 5.6 ! 65, 65 , -0.17 0.5 2 ! // 11 it 11 65 65 -0.45 ! 1.5 3 I " a 11 11 60 60 -1.4 4.6 4 a 11 it 11 61 60 0 . 0 5 \ a .1 11 it 11 21 70 ° ; 0 ( 7 ) a -acid t'WA Keto glutaric acid Na itffc N H , WX&'jtT I J ffc L T , P j * l i ' J » $ w : L (+ ) & glutamic ffi X V -ii a-Keto glutaric acid Na tii<rj NHs 7k ' l ' i:?•-• 11 &m'/t ( g ) fell tS Raney-Ni (ws) (?*) 5 10 5 iff NI-I.OH (28?*) (ml.) 18 18 40 40 18 (atm.) 80 15 15 80 15 if: ft iU ffi C C ) 50 50 40 40 50 Glutamic acid Ca?ii HC1 V(3'°) + 0.5 + 0.5 + 0.6 +0.4 + 0.45 1.67 1.67 2 1.33 1.5 - 132 -fc*) (39) K re-* e> rose H I H n n. ; r-Trfc(iO"/,', :C-Ji.••-rfe.i-f LA;. ( 1 ) dl-a-oxy glutaric acid diethyl ester Sr Raney-Ni (W5) W r f t T , ESS. zK&TMiT, K * 3 R ^ -WRfcKiSfctfV'. t»W'lj,'fW'l'-c(t L ( - ) w ; , JHHsttffitflM'-Stt DC+) WC=F#feH4* * .1 t o C O « | i « t X ! R l » rt-kcto glutaric acid diethyl ester f)Uv<0«i-&tm b-ZbZtf. P?Htm<, B 8i»:^'>A ' i. '««j . l-:ll ;jn<Jo JK*J8KHH.t 2. 4-dinitro phenyl hydrazine fcJIjVTf&fc'U SB XVI «r f i« +, ++ It a-keto acid Jfe *C*V cyclo hexanc ftS'/^li W t t i fco //J&jttt' SV'j'i t i> OlffthiX -ftti%X£> o ffs x v i v< dl-a oxy glutaric acid diethyl ester »^f :j sF$2ttK{5 *& *£ U it; -ft <t- t s !kl& a-oxy glutaric diethyl es ( g ) di m C O ' i'V mi (ml.) 2.4-DNT •IS. fc. Ca]n P ( * 1 5 2 60 i 5 Hexan 10 -t- o : 0 2 n ti II 80—85 1 I it ll 10 •Ht + ? — 3 it it it 100—120 it it 10 + +o.o5 ; 1.04 4 it II II 140 1 n ll 10 '-H- + 0.2 j 4.08 5 n it II 80 j ti EtOH 10 + -0.3 6.25 6 ti it it 100 II it 10 -0.3 ' 6.25 7 it ti it 120 1 it it 10 + -0.45 9.3 8 n it it 140 it ti 10 - -0.3 | 6.25 9 II it it 160 ; II II 10 - • -0.2 i 4.08 10 it it it 100 II AcoEt 10 -0.15 ' 3 .12 11 it it ti 120 i it n 10 + -0.3 6.25 12 it II it 150 ; II II 10 -0.3 1 6.25 13 II II II ioo : it Dioxan 10 -0.2 4.08 14 it II II 120 ; II it 10 + -0.15 3.12 15 tt it ti 140 i it n 10 -H- -0.3 ! 6.25 m m i. n LXWCMzZ Lv<ti&hhZ> \m<>->Tm#t<r)Ktt£ <> £ izStKSrK*A:. •)m. I \Zti^XUhMz%mz i.WftJ8.-i£i:l&m L, ##3H41*J. d-camphor, d-borneol, 1-borneol & } li(t£-i£, a-keto glutaric acid diethyl ester, a-oximino glutaric acid O diethyl iiiU Na JJiJrjSTCL •C-tW?s5Si|;ov>-CKi,iJ-LA;. ( 1 ) -Jitil I IZis^-Xi&^fjC'm-VX a-keto glutaric acid diethyl ester l i ^ f t S X S U A: J>s Z<D$;?\: fc-HOll L. d-camphor', d-borneol, l-borncol k J (i-i-ViiO) vi S I ; O V - T J j L A; . nuo)t».\&tmnmitim*>mwsi<»wf&n aun^tc «-ox y acid i * m. D(+) t - 133 -142 «H1«0I, Yli-JU Ufl, tt/JittJSi- (40) •*^ffitt:W»:'tt-if/*->' a-keto glutaric acid diethyl ester to'yi:jt i m iiii i»i tt 'iV ft- !ki& oxy acid Xa iS ; (g) ; c w s ) ( * ) | ( g ) (ml.) 7k : * * i rR (ml.) 1 (atm.) t u m Cc) [ajn \ P O O 1 ! • ~w ' | s j L>-camphor 2 — - I 63 80 + 2.1 24.4 2 • ti j 3 ll 2 — — i 60 100 + 0.57' j 6.7 3 n j 3 " 2 — — 60 130 + 0.5 ! 5.84 4 if i 1 i " 2 — — 60 150 + 0.58 j 6.4 5 it j 5 ! " ' 2 Hexan 10 26 100 , -0.6 ; 7 6 ti 5 // 2 ll 20 — j 60 75 -0.6 ! 7 7 ir j // 20 ll 30 — i 38 75 ; -1.3 15.1 8 it 10 j D-Borneol 2 ll 50 — ,' io 100 -0.1 1.1 9 it j 10 i I.-Borneol 2 AcoEt 50 — j 10 100 -0.05 0.56 10 it 1 1 0 i D-Uorneol 2 ll 50 — 1 10 SO -0.1 1.1 11 it i 10 ! L~Borneol 2 ll 50 — 10 80 -0.1 1.1 12 ii 5 ! D~C'amphor2 ll 10 , — 15.5 85 j -0.6 7 13 II 5 , D-Camphor2 ll 20 ' - 15.5 85 -0.9 10 14 ti i 1 0 , D~Camphor5 II 20 — 59 80 -0.4 +•7 IS it 10 L-lJorneol 2 — — 60 80 i + 2 23.3 16 i io . D-Borncol 2 — — 60 80 ; -0.6 7 17 j 5 ; D~Camphor5 Etanol 40 6 60 50 : + 0.6 7 ( 2 ) MiSkin (5),(6) IZtsi'ti) V-& r-i'r'Slt-f-JI- k I't-IU U a-oximino glutaric acid O diethyl ester &XV Na fjiSrMMfiC d-camphor, d-borneol, l-borneol lOJIj/FKiCSyi; U 'C -C^S iWlOV 'X^ff. L/-. \.*-ft\.to\m.-KMlfHZH^Xi> m~.1i<nVi<fXU L ( + ) ">\ (S ft'Cli D ( - ) ";!« glutamic acid -ft-Jfcf5'?lLQft<n:}%'4!-~Y a-oximino glut uric 3cid (?) diethyl ester N'a i^t^iii'/ii tt Uarrey-Ni ( g ) 1 ( W 7 ) 0 0 i * tt (ml.) t i l ( g ) 1"J 'kvKWK (atm.) ill HE $:J& glutamic acid C«3l» H l ' l P C O 1 ' Ester 5 i 10 AcoEt 20 D-Camphor2 20 120 + 0.33 1.1 2 ti- II II // D-Borneol 2 1 it tr -f-0.33 tt 3 i /' lt] II II // I,-Borneol 2 . tt tt -0.5 - 1.6 4 >5 ! 5 // D-Borncol 2 tt 60 + 0.1 0.3 5 ! /// ' 5 < // L-Horncol 2 it it -0.5* 1.7 LCO Ut> - 134 -Production of a-hydroxyglutaric acid diethyl ester, I; Na sa l t , X j Glutamic acid, 0 47 Experiments Total +157$ 25 Expts. 71 Expts. -32$ - 10 I X X X X 143 Expts. Total 157 - 329 = -172$ Average = -1.20$ + 0.4$1 - 20 I I x 1 _?0V2 _ Q.3S7 - 135 -Table X. Expts. 20 - 37 Reduction of a-ketoglutaric acid Na salt i n aqueous soln. at pH 8 Table X: I P $ - 136 -Table XI: X Table XII: 0 + 11 I I + 10 + 9 + 8 + 7 I I + 6 I + 5 I X + 4 I + 3 I I + 2 I I + 1 I I 0 I I - 1 I X - 2 I I - 3 X X - 4 X 0 - 5 I X - 6 X 0 - 7 X X - 8 - 9 I X - 10 I I -11 • • - 20 I I I Table X + 19 expts. + 81$ 8 expts, - 10 expts. =Ja£ + 20$ 37 expt. * 0.54$ V Total + 20expts. + 86$ 15 expts. - 25 expts. - 144$ - 58$ 59 expts. = -0.98$ + 0.7$ 1 

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