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UBC Theses and Dissertations

Endor and Epr studies of organic radicals in solution Kennedy, David Edward Bertram 1974

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C t ENDOR AND EPR STUDIES OF ORGANIC RADICALS IN SOLUTION by DAVID E.B. KENNEDY B . S c , Queen's U n i v e r s i t y , 1966 A T h e s i s S u b m i t t e d i n t h e Requirements D o c t o r o f P a r t i a l F u l f i l m e n t o f f o r t h e Degree o f P h i l o s o p h y In t h e Department o f C h e m i s t r y 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 BRITISH COLUMBIA, 1974 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Chemistry The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date March 20, 1974 Professor: CA. McDowell Abstract The construction and operation of a high power liquid phase Electron Nuclear Double Resonance (ENDOR) spectrometer is described in detail. The several modifications of existing methods that make this spectrometer unique are emphasized, particularly the problem of>;utilizing very high rf fields without excessive rf interference. Some inexpensive and simple methods that were developed for increasing the strength of the rf field are discussed in detail. The completed spectrometer was used to study very small varia-tions in hyperfine couplings in solutions of the tri-t-butyl phenoxy (TTBP) radical. The theory for the treatment of such ENDOR-measured coup-lings is discussed, and this means was used to determine the existence of very weak hydrogen bond formation in solutions of TTBP and proton-donating solvents. The formation constants and enthalpy of formation for these bonds can be obtained from this theory. The spectrometer was used for an ENDOR study of some common and similar stable free radicals: a,a'-Diphenyl-3-Picryl Hydrazyl (DPPH), Picryl-N-Amino Carbazyl (PAC) and a,Y-Bisdiphenylene-e-Phenyl Allyl (BDPA). Although the Electron Paramagnetic Resonance (EPR) of all of these compounds has been known for years, there has never been a complete interpretation of their EPR spectra. The ENDOR-obtained hyperfine coupling constants gave complete interpretations of these complex EPR spectra. Spectra were only obtained for these radicals when stringent ENDOR conditions were satisfied: a better understanding of these conditions will make liquid ENDOR acless specialized tool. Values for the spin-spin relaxa-tion time T2 could be estimated for solutions of these radicals from the P r o f e s s o r : C A . McDowell ENDOR s p e c t r a . The EPR s i m u l a t i o n s and t h e a s s i g n m e n t o f t h e ENDOR v a l u e s t o g e t h e r y i e l d t h e f i r s t a c c u r a t e s p i n d e n s i t y maps f o r t h e s e t h r e e s p e c i e s . The m o l e c u l a r d i s t o r t i o n t h a t i s i n f e r r e d f r o m t h e ENDOR s p e c t r a must be taken i n t o a c c o u n t i f t h e s e a c c u r a t e v a l u e s a r e t o be used f o r d e t a i l e d m o l e c u l a r s p i n d e n s i t y c a l c u l a t i o n s . F i n a l l y , t h e optimum c o n d i t i o n s mentioned above were used t o o b t a i n t h e ENDOR s p e c t r a o f some r e l a t e d d r u g p r e c u r s o r s : p h e n o x a z i n e , p h e n o t h i a z i n e and c h l o r o p h e n o t h i a z i n e . The ENDOR h y p e r f i n e v a l u e s o f g e n e r a t e d r a d i c a l s o f t h e s e compounds were used t o s i m u l a t e t h e i r EPR s p e c t r a . A novel c h l o r i n e h y p e r f i n e c o u p l i n g c o n s t a n t was measured by a c a r e f u l s t u d y o f t h e s e s i m u l a -t i o n s . C h l o r i n e c o u p l i n g s have r a r e l y been o b s e r v e d p r e v i o u s l y i n a r o m a t i c f r e e r a d i c a l s i n s o l u t i o n . The a c c u r a t e ENDOR p r o t o n c o u p l i n g s and a s s i g n -ment were f i r s t r e q u i r e d i n o r d e r t o o b t a i n t h i s s m a l l and unusual c h l o r i n e c o u p l i n g . i v TABLE OF CONTENTS Page A b s t r a c t i i L i s t o f T a b l e s v i i L i s t o f F i g u r e s . •• v i i i Acknowledgements x i v C h a p t e r s : One: I n t r o d u c t i o n 1 Two: T h e o r e t i c a l 11 2.1 T r a n s i t i o n F r e q u e n c i e s 12 2.2 P h e n o m e n o l o g i c a l T r e a t m e n t 15 2.3 The H y p e r f i n e C o u p l i n g C o n s t a n t C o r r e c t i o n o f the Enhancement 27 2.4 T h e o r e t i c a l Requirements f o r H 2 30 2.5 D e t e c t i o n Methods 32 2.6 S p e c t r o m e t e r Requirements 35 2.7 T r e a t m e n t o f Data 38 2.8 S c a t c h a r d T h e o r y 40 T a b l e o f C o n t e n t s c o n t i n u e d v Page T h r e e : E x p e r i m e n t a l 45 3.1 High-Power S p e c t r o m e t e r 47 3.2 The C a v i t y .. 47 3.3 I n t e r n a l F i e l d Measurements... 52 3.4 R a d i o - F r e q u e n c y System 55 3.5 Servo System and C a p a c i t o r 57 3.6 Microwave and D e t e c t i o n System 60 3.7 Magnet and S h i e l d i n g 61 3.8 Temperature C o n t r o l 62 3.9 EPR D e t e c t i o n 63 3.10 ENDOR O p e r a t i o n 64 3.11 Sample P r e p a r a t i o n (a) T r i - t - b u t y l Phenoxy 69 (b) S t a b l e F r e e R a d i c a l s 70 (c) G e n e r a t e d F r e e R a d i c a l s 72 Four: R e s u l t s and D i s c u s s i o n 74 4.1 The Phenoxy R a d i c a l and Hydrogen Bonding 75 4.2 a , a ' - D i p h e n y l - B - P i c r y l H y d r a z y l (DPPH) 88 4.3 P i c r y l - N - A m i n o C a r b a z y l (PAC) I l l 4.4 a,Y-B1s'diphenylene-B-Phenyl A l l y ! (BDPA) 123 4.5 G e n e r a t e d F r e e R a d i c a l s 137 (a) P h e n o x a z i n e 138 (b) P h e n o t h i a z i n e 141 (c) 2 - C h l o r o p h e n o t h i a z i n e 145 v i T a b l e o f C o n t e n t s c o n t i n u e d Page R e f e r e n c e s 152 . v i i LIST OF TABLES T a b l e Page 1. S t r u c t u r e and R e l a t i o n s h i p o f M o l e c u l e s S t u d i e d by ENDOR 7 2. C a v i t y Q u a l i t y - F a c t o r Measurements 53 3. Measurements o f t h e Rf F i e l d a t V a r i o u s Input V o l t a g e s 54 4. The ENDOR-Determined F o r m a t i o n C o n s t a n t s and E n t h a l p y o f For m a t i o n o f Hydrogen Bonds i n CHC1 3 - TTBP s o l u t i o n 86 5. H y p e r f i n e C o u p l i n g s and S p i n D e n s i t i e s o b t a i n e d f o r DPPH by ENDOR and EPR 94 6. A c c i d e n t a l O v e r l a p s i n DPPH C o u p l i n g C o n s t a n t s 109 7. H y p e r f i n e C o u p l i n g s and S p i n D e n s i t i e s O b t a i n e d f o r PAC S o l u t i o n s from ENDOR and EPR VI6 8. Some A c c i d e n t a l O v e r l a p s i n PAC C o n s t a n t s 121 9. H y p e r f i n e C o u p l i n g s , S p i n D e n s i t i e s and 1^ V a l u e s o b t a i n e d f o r BDPA by ENDOR 127 10. H y p e r f i n e C o u p l i n g s o f Ge n e r a t e d R a d i c a l s O b t a i n e d by ENDOR and EPR 143 v i i i LIST OF FIGURES F i g u r e Page 1. Energy L e v e l diagram f o r a system w i t h S = h and I = h showing EPR and ENDOR t r a n s i t i o n s 16 2. The ENDOR and DNP e x p e r i m e n t s : arrangements o f s o u r c e s and d e t e c t o r s 18 3. C o i l s used f o r low power attempts and a s s o c i a t e d tank c i r c u i t 46 4. G e n e r a l B l o c k Diagram o f X-Band ENDOR and EPR S p e c t r o m e t e r : arrangements o f d e t e c t o r s and f r e q u e n c y g e n e r a t o r s 48 5. Squeezed- r f c o i l s o f c a v i t y . The t e f l o n i n s u l a t o r s and co p p e r r f s h i e l d s a r e a l s o v i s i b l e 51 6. (a) F o u r - t o - o n e r f t r a n s f o r m e r and (b) S c h e m a t i c d i a g r a m o f 16:1 r f t r a n s f o r m e r 56 7. Servo System and c a v i t y m o d i f i c a t i o n s : The c a p a c i t o r and i t s s e r v o d r i v e system ( s h i e l d e d ) a r e on lower l e f t . E x p l o d e d view o f t h e c a v i t y , c o o l i n g system and sample dewar on r i g h t . A 16:1 r f t r a n s f o r m e r i s a l s o shown 59 i x L i s t o f F i g u r e s c o n t i n u e d F i g u r e Page 8. D e t a i l e d B l o c k Diagram o f high-power l i q u i d ENDOR ..Spectrometer, showing a l l component c o n n e c t i o n s 65 9. T y p i c a l ENDOR o f * T r i - t - b u t y l phenoxy i n c a r b o n t e t r a c h l o r i d e (5%) and m i n e r a l o i l a t 20°C. The f r e e p r o t o n f r e q u e n c y and the as s i g n m e n t s o f t h e p r o t o n s a r e shown 76 10. ENDOR o f TTBP i n m i n e r a l o i l (100%) a f t e r a g i n g , showing the appearance o f l i n e s f r o m t h e s e c o n d a r y o x i d a t i o n s p e c i e s 77 11. Temperature v a r i a t i o n o f meta-proton coupTrhg c o n s t a n t i n TTBP. A few o f t h e s o l v e n t c o n c e n t r a t i o n s s t u d i e d a r e shown 82 12. C o n c e n t r a t i o n dependence o f TTBP c o u p l i n g c o n s t a n t a t -30°C w i t h added C C l ^ 9 CHC1 3 and CDC1 3 83 13. S c a t c h a r d P l o t o f meta p r o t o n c o u p l i n g c o n s t a n t o f TTBP a t -39°C i n CHC1 3. The arrov/s i n d i c a t e t h e 6 l i m i t s o f 0.2 and 0.8 85 X L i s t o f F i g u r e s c o n t i n u e d F i g u r e Page 14. The i s o t o p e e f f e c t on t h e hydrogen bond: c o n c e n t r a t i o n dependence o f the d i f f e r e n c e i n meta p r o t o n c o u p l i n g i n TTBP between added CHC1 3 and CDC1 3 87 15. c t j C t ' - P i p h e n y l - B - P i c r y l h y d r a z y l r a d i c a l s t r u c t u r e , showing numbering system and c o u p l i n g s o b t a i n e d (G) 89 16. (a) EPR o f DPPH sample i n m i n e r a l o i l used f o r ENDOR and (b) ENDOR spectrum o f same sample o f DPPH a t room te m p e r a t u r e 92 17. (a) EPR o f (10~ 4M) DPPH i n o u t g a s s e d n-heptane a t 20°C and (b) S i m u l a t e d EPR o f DPPH u s i n g h y p e r f i n e c o u p l i n g s f r o m ENDOR 100 18. S i m u l a t e d ENDOR s p e c t r a o f DPPH u s i n g : (a) DPPH c o u p l i n g s o b t a i n e d i n t h i s work (b) Measured ENDOR s p e c t r u m o f DPPH (c) 1 s t s e t o f DPPH c o u p l i n g s from r e f 80 (d) 2nd s e t o f DPPH c o u p l i n g s from r e f 80 105 x i L i s t o f F i g u r e s c o n t i n u e d F i g u r e Page 19. P i c r y l - n - a m i n o c a r b a z y l r a d i c a l , showing numbering system and a p p r o x i m a t e c o u p l i n g s o b t a i n e d (6) 112 20. EPR o f (10" 3M) PAC i n m i n e r a l o i l as used f o r ENDOR 114 21. (a) ENDOR spectrum o f PAC i n m i n e r a l o i l a t 340K and (b) ENDOR spectrum o f same sample o f PAC a t * 290K. The enlargment shows t h e c e n t r a l l i n e s 115 22. (a) EPR o f (10 _ l ,M) PAC i n o u t g a s s e d C C 1 4 a t 20°C and (b) S i m u l a t e d EPR o f PAC u s i n g t h e h i g h e r t e m p e r a t u r e h y p e r f i n e c o u p l i n g s o b t a i n e d by ENDOR.. 120 23. a . Y - B i s d i p h e n y l e n e - g - P h e n y l a l l y l f r e e r a d i c a l , showing s t r u c t u r e , numbering scheme and a p p r o x i m a t e c o u p l i n g s ( G ) . . . 124 24. (a) EPR o f (10" 3M) BDPA i n m i n e r a l o i l as used f o r ENDOR (b) ENDOR o f same BDPA sample a t 340K 126 25. Low t e m p e r a t u r e ENDOR o f (10" 3M) BDPA i n m i n e r a l o i l a t 300K, showing unusual o u t e r l i n e i n t e n s i t y 130 L i s t o f F i g u r e s c o n t i n u e d F i g u r e Page 26. Enlargement o f c e n t r a l l i n e s o f p r e v i o u s BDPA sp e c t r u m , showing weak phenyl l i n e s 132 27. (a) EPR o f (10 - i fM) BDPA i n o u t g a s s e d benzene a t room t e m p e r a t u r e and (b) S i m u l a t i o n o f EPR u s i n g ENDOR h y p e r f i n e c o n s t a n t s 134 28. Phenoxazine n e u t r a l r a d i c a l ENDOR a t room t e m p e r a t u r e i n m i n e r a l o i l 139 29. (a) EPR o f (10 _ l +M) ph e n o x a z i n e n e u t r a l r a d i c a l i n n-heptane and (b) S i m u l a t i o n o f EPR u s i n g ENDOR c o n s t a n t s 140 30..Room t e m p e r a t u r e ENDOR o f o u t g a s s e d p h e n o t h i a z i n e n e u t r a l r a d i c a l i n m i n e r a l o i l 142 31. (a) EPR o f p h e n o t h i a z i n e n e u t r a l r a d i c a l i n n-heptane a t room t e m p e r a t u r e and (b) S i m u l a t i o n o f EPR u s i n g ENDOR c o n s t a n t s 144 32. ENDOR o f 2 - c h l o r o p h e n o t h i a z i n e n e u t r a l r a d i c a l i n m i n e r a l o i l a t 30°C 146 x i i i L i s t o f F i g u r e s c o n t i n u e d F i g u r e Page 33. (a) EPR o f c h l o r o - p h e n o t h i a z i n e n e u t r a l r a d i c a l i n n-heptane a t room t e m p e r a t u r e and (b) S i m u l a t i o n o f EPR u s i n g ENDOR c o n s t a n t s 148 34. (a) E n l a r g e m e n t o f o u t e r l i n e s o f c h l o r o - p h e n o t h i a z i n e ; (b) S i m u l a t i o n s o f o u t e r l i n e s w i t h c h l o r i n e c o u p l i n g (a^-j = 75 mG) and (c ) S i m u l a t i o n o f o u t e r l i n e s w i t h o u t c h l o r i n e c o u p l i n g 15,0 ACKNOWLEDGEMENTS x i v I w i s h t o thank my r e s e a r c h d i r e c t o r , P r o f e s s o r C A . McDowell, f o r h i s h e l p and g u i d a n c e d u r i n g my s t u d i e s a t U.B.C, e s D e c i a l l y d u r i n g t h e d i f f i c u l t p e r i o d s o f no r e s u l t s . Most s i n c e r e thanks go t o my c o l l e a g u e , Dr. N.S. D a l a i , who a s s i s t e d i n the s p e c t r o m e t e r c o n s t r u c t i o n w i t h u n t i r i n g d e v o t i o n , was always r e a d y w i t h a c r y p t i c comment o r a l o n g d i s c u s s i o n a t any s t a g e o f t h i s r e s e a r c h , and never f a i l e d t o see t h e p h y s i c s b e h i n d t h e c h e m i s t r y we were s t u d y i n g . I am v e r y g r a t e f u l t o Dr. R.D. A l l e n d o e r f e r , SUNYAB, f o r h e l p f u l l e t t e r s on s p e c t r o m e t e r d e s i g n d u r i n g t h e e a r l y s t a g e s o f t h i s work and f o r s e v e r a l days o f d i s c u s s i o n d u r i n g a v i s i t t o B u f f a l o . Many o f t h e unus u a l f e a t u r e s o f t h e s p e c t r o m e t e r a r o s e f r o m t h e s e d i s c u s s i o n s . Acknowledgement i s a l s o made o f h i s copy o f SESRS, which was m o d i f i e d d u r i n g t h i s s t u d y f o r l a r g e - m o l e c u l e s p e c t r a l s i m u l a t i o n . J . S a l l o s and T. Markus were i n v a l u a b l e a s s i s t a n t s d u r i n g the c o n s t r u c t i o n , and I wis h t o thank them and t h e e n t i r e s t a f f s o f t h e E l e c t r o n i c and M e c h a n i c a l shops a t U.B.C. f o r so c h e e r f u l l y c o m p l y i n g w i t h e v e r y s t r a n g e r e q u e s t d u r i n g t h e s p e c t r o m e t e r c o n s t r u c t i o n . P r o f e s s o r McDowell and I b o t h acknowledge N.R.C. f o r generous equipment g r a n t s ; I wis h t o a l s o thank them a l o n g w i t h t h e U.B.C. C h e m i s t r y department f o r g r a d u a t e s c h o l a r s h i p s and a s s i s t a n t s h i p s . F i n a l l y , I would l i k e t o thank my w i f e , C o l l e e n , f o r her c o n t i n u i n g p a t i e n c e and f o r t y p i n g part, o f t h e o r i g i n a l m a n u s c r i p t . 1 CHAPTER ONE INTRODUCTION The E l e c t r o n N u c l e a r Double Resonance t e c h n i q u e (ENDOR), f i r s t i n t r o d u c e d i n 1957 by Feher] e n a b l e s one t o s t u d y n u c l e a r t r a n s i -t i o n s by means o f E l e c t r o n S p i n Resonance (EPR). T h i s p o w e r f u l d o u b l e r e s o n a n c e t e c h n i q u e has t h e combined s e n s i t i v i t y o f EPR w i t h t h e h i g h r e s o l u t i o n o f NMR. ENDOR o f low t e m p e r a t u r e s o l i d s has been s t u d i e d 2 e x t e n s i v e l y by many workers i n t h i s l a b and e l s e w h e r e . ENDOR o f s o l u t i o n s was n o t o b s e r v e d u n t i l much l a t e r when 3 C e d e r q u i s t o b t a i n e d s i g n a l s f r o m metal-ammonia s o l u t i o n s . In t h e f i r s t ENDOR e x p e r i m e n t using^a-n o r g a n i c s o l v e n t , Hyde and M a k i 4 o b s e r v e d s i g n a l s f r o m C o p p i n g e r ' s r a d i c a l i n n-heptane j u s t above i t s f r e e z i n g p o i n t . S i n c e t h i s t i m e a c o m m e r c i a l l y a v a i l a b l e s p e c t r o m e t e r and s e v e r a l r e s e a r c h groups have f i r m l y e s t a b l i s h e d s o l u t i o n ENDOR as a powerful t e c h n i q u e f o r d e t a i l e d a n a l y s i s o f s t a b l e f r e e o r g a n i c r a d i c a l s i n s o l u t i o n . Hyde 2 c has used t h e t e c h n i q u e t o s t u d y a number o f C o p p i n g e r - r a d i c a l a n a l o g u e s , 6 7 t r i p h e n y l methyl r a d i c a l s , ' and has r e p o r t e d s e v e r a l s t u d i e s on d i f f e r e n t e x p e r i m e n t a l methods. Maki and A l l e n d o e r f e r have s t u d i e d s e v e r a l t r i a r y l and 8 9 t r i p h e n y l methyl r a d i c a l s ' and have a l m o s t i m p o r t a n t p h e n o m e n o l o g i c a l s t u d y o f a phenoxy r a d i c a l . ^ A l l e n d o e r f e r and co-workers have s t u d i e d s p i n - l a b e l l e d 11 12 13 14 compounds, a m a l o n i t r i l e ' d i h y d r o p l e i a d e n e s , p y r r y l r a d i c a l s , hexa-15 16 h e l i c e n e and o t h e r phenoxy. A t h e r t o n e t a l have s t u d i e d s e v e r a l phenoxyl 17 18 19 r a d i c a l s and s e m i q u i n o n e s . Mobius and D i n s e have l o o k e d a t r a d i c a l s i n l i q u i d c r y s t a l s , s t u d i e d c o h e r e n c e e f f e c t s and o b t a i n e d q u a d r u p o l e measure-ments i n s o l u t i o n . Recent r e v i e w s o f t h e e n t i r e l i q u i d ENDOR l i t e r a t u r e have 20 been done by Mobius e t a l ( i n d e x e d by compound) and more c o m p l e t e l y by A t h e r t o n . 2 1 T h i s v e r y b r i e f s u r v e y shows t h a t s o l u t i o n ENDOR has wide a p p l i c a t i o n t o many d i f f e r e n t m o l e c u l a r t y p e s . The u s e f u l n e s s i s o b v i o u s i f one l o o k s c a r e f u l l y a t t h e m o l e c u l e s t h a t have been s t u d i e d : l a r g e , low symmetry m o l e c u l e s w i t h v e r y c o m p l i c a t e d EPR s p e c t r a . The number o f l i n e s i n a s o l u t i o n EPR spectrum i n c r e a s e s i n a m u l t i p l i c a t i v e way so t h a t t h e m o l e c u l e s w i t h more th a n f i v e t y p e s o f n o n - e q u i v a l e n t p r o t o n s can g i v e v e r y complex s p e c t r a . The ENDOR spectrum o f such a system c o n t a i n s t h e same i n f o r m a t i o n w i t h o n l y 2 l i n e s per p r o t o n t y p e . T h e r e i s no i n t e r a c t i o n o f t h e c o u p l i n g s : t h e s i m p l i f i c a t i o n o f t h e spectrum e n a b l e s one t o s t u d y v e r y complex systems w i t h o u t m u l t i p l e o v e r l a p o f s p e c t r a l l i n e s . The NMR t e c h n i q u e on n e u t r a l systems does n o t have t h i s l i m i t a t i o n . NMR on r a d i c a l systems i s becoming more common as new i n s t r u m e n t a l improvements a r e made ( e . g . F o u r i e r T r a n s f o r m t e c h n i q u e s ) . However, NMR on r a d i c a l s s u f f e r s f r o m two drawbacks which make t h e ENDOR t e c h n i q u e a s t r o n g c o m p e t i t o r . F i r s t , 3 t h e s e n s i t i v i t y o f t h e NMR e x p e r i m e n t i s so low t h a t v e r y c o n c e n t r a t e d s o l u -t i o n s a r e n e c e s s a r y . The s p i n - s p i n i n t e r a c t i o n and r e l a x a t i o n i n such s o l u -t i o n s can d i s t o r t t h e system under s t u d y t o such a degree t h a t i n f o r m a t i o n on some t i m e s c a l e s i s l o s t . C o n v e r s e l y , i f a d i l u t e p a r a m a g n e t i c s y s t e m i s s t u d i e d , i t i s common i n NMR t o c h e m i c a l l y enhance t h e weak s p e c t r a , u s u a l l y by a d d i n g a s p i n l a b e l , e.g. DBNO. T h i s masks s m a l l c o u p l i n g s under t h e enhancement l i n e . ENDOR can s t u d y s m a l l c o u p l i n g s i n d i l u t e s o l u t i o n s w i t h none o f t h e s e drawbacks. The second problem w i t h NMR i s t h a t t h e l i n e w i d t h o f a r a d i c a l NMR l i n e s v a r i e s w i t h t h e squa r e o f t h e c o u p l i n g . C o u p l i n g s above a few Gauss (EPR s c a l e ) have l i n e w i d t h s o f s e v e r a l thousand c y c l e s and do n o t g i v e an NMR sp e c t r u m . ENDOR can e a s i l y be used f o r s t u d y o f l a r g e c o u p l i n g s , a l t h o u g h t h e s e a r e n o r m a l l y q u i t e v i s i b l e i n t h e EPR s p e c t r a . T h i s e x p l a i n s t h e r e c e n t i n t e r e s t i n t h e ENDOR t e c h n i q u e : t h e r e s o l u t i o n i s as good as NMR, and t h e s e n s i t i v i t y a l m o s t t h a t o f EPR. T h e r e a r e drawbacks t o t h e ENDOR t e c h n i q u e s which n o r m a l l y a r i s e from t h e p r e s e n t i n s t r u m e n t a t i o n . The s o u r c e o f t h e s e l i m i t a t i o n s and t h e methods f o r o v e r -coming them w i l l be e x p l a i n e d i n d e t a i l i n t h i s work. The o b j e c t o f t h i s s t u d y was t o c o n s t r u c t an ENDOR s p e c t r o m e t e r around an a v a i l a b l e EPR s p e c t r o m e t e r . The e n g i n e e r i n g o f t h i s s p e c t r o m e t e r w i l l be ke p t s e p a r a t e f r o m t h e c h e m i s t r y o f t h e systems under s t u d y . With t h e co m p l e t e d s p e c t r o m e t e r s e v e r a l r e l a t e d r a d i c a l s were s t u d i e d . S e v e r a l o f t h e s e r a d i c a l s a r e v e r y common and i t i s r e m a r k a b l e t h a t t h e ENDOR s p e c t r a o f none have been o b t a i n e d by o t h e r w o r k e r s . The f i r s t s e c t i o n w i l l i n c l u d e t h e n e c e s s a r y t h e o r y t o i n t e r p r e t ENDOR s p e c t r a . I t i s f e l t t h a t t h e t h e o r y f o r EPR has been c o v e r e d 4 a d e q u a t e l y b e f o r e and o n l y t h e i n f o r m a t i o n n e c e s s a r y f o r t h e ENDOR c o u p l i n g s i s i n c l u d e d . Other parameters can be e x t r a c t e d from ENDOR such as t h e s p i n - s p i n r e l a x a t i o n time T 2 and t h e d e r i v a t i o n s f o r t h e s e w i l l be c o v e r e d . S e v e r a l e x p e r i m e n t a l and i n s t r u m e n t a l p arameters were chosen based on t h e above d e s c r i p t i o n . A t r e a t m e n t f o r u s i n g changes i n h y p e r f i n e c o u p l i n g s t o d e t e c t complex f o r m a t i o n i n s o l u t i o n i s a l s o i n c l u d e d . The next s e c t i o n i s a d e t a i l e d d e s c r i p t i o n o f t h e s p e c t r o m e t e r . S e v e r a l u n i q u e f e a t u r e s w i l l be f u l l y d e s c r i b e d , and t h e s t a t e - o f - t h e - a r t i n s t r u m e n t a t i o n and the h i g h r f power m o d i f i c a t i o n s w i l l be d i s c u s s e d . S e v e r a l workers have been u n a b l e t o o b t a i n ENDOR on some o f t h e common r a d i c a l s s t u d i e d , so sample p r e p a r a t i o n w i l l be d i s c u s s e d i n d e t a i l . The c h e m i c a l s t u d i e s w i t h t h e completed s p e c t r o m e t e r f a l l i n t o t h r e e c l a s s e s . F i r s t , ENDOR was used t o s t u d y hydrogen bond complex forma-t i o n i n v a r i o u s s o l v e n t s . Second, s e v e r a l s t a b l e f r e e r a d i c a l s whose EPR i n t e r p r e t a t i o n i s d o u b t f u l were s t u d i e d . F i n a l l y , some b i o l o g i c a l l y a c t i v e m o l e c u l e s (which a r e a n a l o g u e s o f t h e s t a b l e f r e e r a d i c a l s i n the second p a r t ) were made i n t o r a d i c a l s , and t h e ENDOR s p e c t r a o b t a i n e d . I n i t i a l s t u d i e s were c o n d u c t e d on a r a d i c a l t h a t has a l r e a d y been v e r y w e l l s t u d i e d phenomenologica l l y by ENDOR, 1 0 t h e t r i - t e r t - b u t y l phenoxy r a d i c a l (TTBP). S i n c e t h e c o n d i t i o n s f o r o b t a i n i n g ENDOR s p e c t r a f o r t h i s s p e c i e s a r e so v e r y w e l l known, i t was hoped t h a t t h i s sample c o u l d s e r v e as an i n s t r u m e n t a l i n t e n s i t y - s t a n d a r d . A l t h o u g h t h e s e n s i t i v i t y o f t h e ENDOR e x p e r i m e n t i s b e t t e r than NMR, t h e s i g n a l s a r e o n l y about 1% o f t h e EPR i n t e n s i t y . The problem w i t h a newly c o n s t r u c t e d s p e c t r o m e t e r o p e r a t i n g on a d o u b l e r e s o n a n c e p r i n c i p l e i s t h a t i t i s d i f f i c u l t t o t e l l 5 ( i n t h e c a s e o f no o b s e r v e d s i g n a l s ) i f one o f the many components o r th e sample i s r e s p o n s i b l e f o r any f a i l u r e . The c o n d i t i o n s f o r ENDOR o p e r a t e o v e r a narrow range o f s o l v e n t v i s c o s i t y and t e m p e r a t u r e ; f u r t h e r m o r e , not a l l r a d i c a l s d e t e c t a b l e by EPR g i v e ENDOR. The ENDOR enhancement a l s o v a r i e s w i d e l y from m o l e c u l e t o m o l e c u l e and i n t h e b e s t c a s e i s o n l y about 1 0 ~ 1 9 Watt. Hence a s t a b l e sample w i t h a known enhancement and s p e c t r a l c o n d i t i o n s i s d e s i r a b l e . I t was found t h a t TTBP r a d i c a l decays i n a few weeks t o form a new s p e c i e s t h a t i s , d e t e c t a b l e o n l y by ENDOR: t h i s makes i t a v e r y poor i n t e n s i t y s t a n d a r d . However, a d e t a i l e d s t u d y r e v e a l e d t h a t TTBP forms a complex w i t h p r o t o n - d o n a t i n g s o l v e n t s . The e f f e c t o f t h i s hydrogen bond f o r m a t i o n i s so s m a l l t h a t i t i s d o u b t f u l i f i t c o u l d be d e t e c t e d w i t h o u t ENDOR: based upon EPR s t u d i e s o f p r e v i o u s workers i t has been m a i n t a i n e d t h a t TTBP does not form hydrogen bonds. A f o r m a t i o n c o n s t a n t and e n t h a l p y o f f o r m a t i o n were o b t a i n e d from a S c a t c h a r d t r e a t m e n t o f th e ENDOR c o u p l i n g s . T h i s t r e a t m e n t showed t h a t a weak hydrogen bond was b e i n g d e t e c t e d by ENDOR, and g i v e s as w e l l t h e e q u i l i b r i u m l i m i t s f o r t h e weak complex f o r m a t i o n . Data near t h e ends o f t h e e q u i l i b r i u m range have v e r y l a r g e e r r o r s and can thus be e l i m i n a t e d by t h i s method. A l t h o u g h 2 hydrogen bonds have o f t e n been d e t e c t e d i n s o l i d s by ENDOR, t h i s i s t h e f i r s t i n s t a n c e o f hydrogen bond d e t e c t i o n w i t h l i q u i d ENDOR. The next s e c t i o n o f t h i s s t u d y i s the i n t e r p r e t a t i o n o f a l l t h e h y p e r f i n e i n t e r a c t i o n s i n t h e s t a b l e f r e e r a d i c a l a , a'-Diphenyl-B-P i c r y l h y d r a z y l (DPPH). T h i s r a d i c a l has been s t u d i e d f o r s e v e r a l y e a r s w i t h o u t complete i n t e r p r e t a t i o n by any group o f w o r k e r s . I t has p r o b a b l y been used as a f i e l d s t a n d a r d i n a l m o s t e v e r y EPR i n s t r u m e n t . The p r o t o n 6 ENDOR shows t h a t t he u n p a i r e d e l e c t r o n i s c o m p l e t e l y d e l o c a l i z e d o v e r t h e m o l e c u l e . I d e n t i f i c a t i o n o f t h e c o u p l i n g s w i t h t h e p r o t o n s was completed by f i t t i n g t h e EPR, by comparison t o NMR s t u d i e s and by a n a l o g y to o t h e r w e l l r e s o l v e d m o l e c u l e s . The c o n s t a n t s o b t a i n e d show t h a t DPPH i s s l i g h t l y d i s t o r t e d i n s o l u t i o n . The a c c u r a c y o f the ENDOR c o u p l i n g s p r o v i d e s an e x c e l l e n t s t a r t i n g p o i n t f o r any a c c u r a t e t h e o r e t i c a l c a l c u l a t i o n s on t h i s r a d i c a l . A l l o f t h e subsequent m o l e c u l e s s t u d i e d a r e an a l o g u e s o f DPPH and a r e l i s t e d i n T a b l e 1. The next most i m p o r t a n t r a d i c a l s , p i c r y l - n -a m i n o c a r b a z y l (PAC) and a . y - b i s d i p h e n y l e n e - B - p h e n y l a l l y l (BDPA) a r e much more r i g i d t h a n DPPH (see T a b l e 1) y e t show more d i s t o r t i o n i n s s o l u t i o n . A g a i n , no com p l e t e a n a l y s i s e x i s t s f o r e i t h e r o f t h e s e common r a d i c a l s . Both o f t h e s e systems show t e m p e r a t u r e changes t h a t i n d i c a t e m o l e c u l a r d i s t o r t i o n . In PAC t h e c a r b a z y l s i d e o f t h e m o l e c u l e must be bent a t low t e m p e r a t u r e s as i n d i c a t e d by the n o n - e q u i v a l e n c e v i s i b l e i n t h e ENDOR; t h e p i c r y l r i n g must a l s o be r o t a t i n g r a p i d l y on t h e ENDOR t i m e s c a l e . In BDPA t h e r e i s l e s s d i s t o r t i o n v i s i b l e , but t h e u n p a i r e d e l e c t r o n i s d e l o c a l i z e d o v e r t h e phenyl r i n g as w e l l , i n c o n t r a s t t o t h e r e s u l t s o f p r e v i o u s w o r k e r s . The measurements o f T 2 v a l u e s from ENDOR f o r t h e s e m o l e c u l e s can be compared t o T 2 times measured on t h e s e same systems by more c o n v e n t i o n a l methods. F i n a l l y , t h e a s s i g n m e n t o f the c o u p l i n g s t o s p e c i f i c p r o t o n s i n t h e s e m o l e c u l e s s e r v e s as an e x c e l l e n t check on t h e as s i g n m e n t s i n DPPH. PAC and DPPH a r e a l m o s t i d e n t i c a l and a r e m u t u a l l y c o n s i s t e n t i n t h e i r ENDOR. BDPA has no p i c r y l r i n g , and t h e c o u p l i n g s i n DPPH a s s i g n e d here a r e m i s s i n g i n t h e BDPA spectr u m . 7 Table 1. S t r u c t u r e and r e l a t i o n s h i p o f M o l e c u l e s S t u d i e d by ENDOR NAME X Z X Y Y DPPH N NO BONDING l-PICRYL PAC N BOND N-PICRYL PHENOXAZINE N 0 PHENOTHIAZINE N 2-CHLORO PHENOTHIAZINE N BDPA PHENARSAZINE N BOND AS-CL PHENYL DIPHENYLENE IMINO DIBENZYL N* •c-c< 8 The f i n a l p a r t o f t h i s s t u d y i s on more d i s t a n t a n a l o g u e s , p h e n o t h i a z i n e and phe n o x a z i n e compounds. These a r e t h e most i n t e r e s t i n g compounds f o r d e m o n s t r a t i n g . f u r t h e r a p p l i c a t i o n s o f t h e ENDOR t e c h n i q u e . L i q u i d ENDOR i s one o f t h e few ways a v a i l a b l e t o measure t he e l e c t r o n n u c l e a r h y p e r f i n e i n t e r a c t i o n i n .a l a r g e m o l e c u l e and t h i s may be o f c o n s i d e r a b l e i m p o r t a n c e w i t h m o l e c u l e s o f b i o l o g i c a l i n t e r e s t . For one t h i n g , m a c r o m o l e c u l a r systems w i l l o f t e n h i n d e r t h e f r e e r o t a t i o n o f a s p i n - l a b e l l e d o r r a d i c a l s p e c i e s . Thi 'S w i l l e f f e c t i v e l y mask hyper-f i n e s t r u c t u r e v i s i b l e i n EPR. However, t h e s e same systems a r e o f t e n l a r g e enough t o nroduce many t y p e s o f r a d i c a l systems which w i l l not e a s i l y d e cay. S o l i d s t a t e s t u d i e s a r e o f t e n f r u s t r a t e d by the d i f f i c u l t y o f growing a c c e p t a b l e s i n g l e c r y s t a l s o f a m a c r o m o l e c u l e . M o l e c u l e s above a c e r t a i n s i z e w i t h enough f u s e d r i n g s w i l l s t a b i l i z e s e v e r a l t y p e s o f r a d i c a l i n t e r m e d i a t e s : c a t i o n and a n i o n , and n e u t r a l from s i d e - c h a i n r u p t u r e . T h e r e i s abundant e v i d e n c e t h a t f r e e r a d i c a l s o f t h e s e systems a r e e s s e n t i a l b i o l o g i c a l i n t e r m e d i a t e s . P h e n o t h i a z i n e and phe n o x a z i n e a r e t h e p r e c u r s o r s o f l a r g e f a m i l i e s o f n e u r o l e p t i c s and dyes , a l l b i o l o g i c a l l y a c t i v e . The l i t e r a -t u r e l i s t s s e v e r a l EPR s t u d i e s o f r a d i c a l s o f t h e s e s e r i e s . A l m o s t a l l o f t h e members w i t h s e v e r a l s i d e c h a i n s show no EPR r e s o l u t i o n : o n l y 4, 5 o r 7 broad l i n e s . ENDOR i s w e l l s u i t e d t o a s t u d y o f such u n r e s o l v e d s p e c i e s . The major problem, based on t h e p a r e n t s o f the s e r i e s i s s t a b i l i z a t i o n o f r a d i c a l s i n s u f f i c i e n t c o n c e n t r a t i o n t o see ENDOR. These s p e c i e s a l s o show t h e c o r r e c t n e s s o f the measured c o u p l i n g s f o r DPPH, PAC and BDPA. Recent work by Gubanov e t a l wifchia F o u r i e r T r a n s f o r m t e c h n i q u e gave a s y n t h e t i c EPR spectrum o f DPPH. The c o n s t a n t s so o b t a i n e d do not a g r e e w i t h t h e e x p e r i m e n t a l l y measured ENDOR c o n s t a n t s ; 9 t h e c o m p l e x i t y o f t h e DPPH EPR spectrum i n d i c a t e s t h a t s e v e r a l d i f f e r e n t s e t s o f c o n s t a n t s might s i m u l a t e t h e spe c t r u m , and t h e argument c o u l d be made t h a t t h e ENDOR c o n s t a n t s do n o t a p p l y t o the EPR spectrum. Pheno-t h i a z i n e and phenoxazine show good EPR r e s o l u t i o n i n the wings o f t h e s p e c t r a . The m i n e r a l o i l ENDOR c o n s t a n t s gave an e x c e l l e n t f i t to the EPR i n n-heptane. I t can be argued t h a t s i n c e t h e c o n d i t i o n s f o r DPPH were i d e n t i c a l , t h e c o n s t a n t s s h o u l d be as a c c u r a t e . S i n c e some doubts were r a i s e d , t h e F o u r i e r t r a n s f o r m c o n s t a n t s were f u r t h e r checked by s i m u l a t i n g . t h e ENDOR spe c t r u m , knowing t h e number o f p r o t o n s f o r each l i n e and e s t i -m a t i n g T 2 c o r r e c t i o n s from t h e EPR. Such s i m u l a t i o n ^ p r o v e s c o n c l u s i v e l y t h e c o r r e c t n e s s o f t h e s e v a l u e s : t h e number o f s i m u l a t e d ENDOR l i n e s does n o t a g r e e w i t h t h e e x p e r i m e n t a l ENDOR spectru m . A s p e c i a l t e c h n i q u e f o r o b t a i n i n g t h e r a d i c a l s o f t h e s e b i o l o g i c a l systems i n m i n e r a l o i l i s de s c r i b e d . . . . S e v e r a l o f t h e dyes t r i e d gave no r a d i c a l by t h i s method, b ut f u r t h e r work i s i n p r o g r e s s . The l i m i t i n g f a c t o r does not seem t o be t h e p r e p a r a t i o n o f the r a d i c a l , but t h e v e r y low s o l u b i l i t y o f t h e s e r a d i c a l s i n m i n e r a l o i l , c o u p l e d w i t h t h e d i f f i c u l t i e s o f removing a l l d i s s o l v e d oxygen from t h i s s o l v e n t . F i n a l l y , an i n d i r e c t measurement o f a c h l o r i n e c o u p l i n g i s p o s s i b l e i n c h l o r o p h e n o t h i a z i n e . T h i s i s o f i n t e r e s t as c h l o r i n e c o u p l i n g s a r e v e r y r a r e l y seen i n s o l u t i o n EPR work, e s p e c i a l l y i n l a r g e a r o m a t i c p l a n a r r a d i c a l s i n s o l u t i o n . A l t h o u g h t h e c h l o r i n e ENDOR l i n e s l i e o u t s i d e t h e range o f t h e s p e c t r o m e t e r , t h e a c c u r a t e p r o t o n c o u p l i n g s o b t a i n e d by ENDOR must c e r t a i n l y a c c u r a t e l y p r e d i c t t h e EPR spectrum as shown above. When the v a l u e s f o r c h l o r o p h e n o t h i a z i n e f a i l e d t o p r e d i c t s h o u l d e r s and smal l l i n e shape v a r i a t i o n s , v a r i o u s c h l o r i n e v a l u e s were added t o t h e 10 s p e c t r u m . T h i s p r o c e d u r e gave i n d i r e c t l y t h e c h l o r i n e c o u p l i n g c o n s t a n t . The v a l u e o b t a i n e d was equal t o t h e minimum t h e o r e t i c a l l i n e w i d t h . Such a s m a l l v a l u e c o u l d n ot be measured d i r e c t l y s v and even i n d i r e c t l y , i t i s n e c e s s a r y t o know a c c u r a t e l y a l l t h e o t h e r v a l u e s from t he ENDOR spectru m . These r e s u l t s e x p l a i n why c h l o r i n e c o u p l i n g s a r e so seldom seen i n s o l u t i o n EPR s t u d i e s . Much o f the a m b i g u i t y i n s t u d i e s o f c h l o r i n e - c o n t a i n i n g systems can now be r e s o l v e d w i t h ENDOR and EPR s t u d i e s . 11 CHAPTER TWO THEORETICAL I n t r o d u c t i o n T h i s s e c t i o n c o n t a i n s a b r i e f o u t l i n e o f t h e t h e o r y n e c e s s a r y f o r i n t e r p r e t a t i o n o f EPR and ENDOR s p e c t r a i n s o l u t i o n . The g e n e r a l 22 23 t h e o r y o f EPR i s c o v e r e d q u i t e a d e q u a t e l y e l s e w h e r e ' and o n l y t h e t r e a t m e n t n e c e s s a r y f o r t h e e f f e c t s u n i q u e t o ENDOR w i l l be i n c l u d e d h e r e . A b r i e f d i s c u s s i o n o f t h e t h e o r e t i c a l r e q u i r e m e n t s f o r an ENDOR s p e c t r o -meter w i l l f o l l o w t h e d e r i v a t i o n o f t h e s i z e o f t h e ENDOR e f f e c t i n room t e m p e r a t u r e s o l u t i o n s . F o r c o m p l e t e n e s s , t h e d e r i v a t i o n o f t h e S c a t c h a r d p l o t f o r a t r e a t m e n t o f ENDOR c o u p l i n g s i s a l s o i n c l u d e d . T h i s w i l l be n e c e s s a r y f o r t h e d i s c u s s i o n on.hydrogen b o n d i n g . . 12 2.1 T r a n s i t i o n F r e q u e n c i e s I f a m o l e c u l a r system c o n t a i n i n g an u n p a i r e d e l e c t r o n i s p l a c e d i n a s t r o n g m a g n e t i c f i e l d , H , the de g e n e r a c y o f the ground s t a t e o f t h i s system i s removed and the l e v e l s i n the s t a t e undergo a Zeeman s p l i t t i n g . I f t h i s system c o n t a i n s n u c l e i w i t h m a g n e t i c n u c l e a r s p i n s , I , the l e v e l s a r e f u r t h e r s p l i t , f i r s t by t h e n u c l e a r Zeeman terms as t h e n u c l e i i n t e r a c t w i t h t h e magnetic f i e l d , and s e c o n d l y by the h y p e r f i n e i n t e r a c t i o n , as t h e n u c l e i i n t e r a c t w i t h t h e u n p a i r e d e l e c t r o n . For a s i m p l e m o l e c u l a r system c o n t a i n i n g o n l y 1 u n p a i r e d e l e c -t r o n , S = h,and o n l y one p r o t o n , I = %, t h e o r d e r i n g o f the l e v e l s i s as shown i n F i g 1. I f t h e s e l e v e l s a r e s u b j e c t e d t o h i g h f r e q u e n c y f i e l d s o f v a r i o u s v a l u e s , t r a n s i t i o n s may by i n d u c e d between t h e l e v e l s . The a b s o r p t i o n o f en e r g y as t h e magnetic f i e l d i s swept g i v e s r i s e t o EPR o r NMR l i n e s , d e p e n d i n g upon t h e f r e q u e n c y o f t h e i r r a d i a t i o n f i e l d . These i r r a d i a t i o n f i e l d s a r e l a b e l e d Hl (microwave) and H 2 (NMR o r r a d i o f r e q u e n c y ) . A number o f s i m p l i f i c a t i o n s f o r the c a s e s s t u d i e d w i l l make t h e d e r i v a t i o n s c l e a r e r . In s o l u t i o n s c o n t a i n i n g f r e e r a d i c a l s , each m o l e c u l e c o n t a i n s a l a r g e number o f n u c l e i , u s u a l l y w i t h s p i n I = h but o n l y one u n p a i r e d e l e c t r o n p e r m o l e c u l e w i t h S = h. The m o l e c u l e s i n s o l u t i o n a r e n o r m a l l y t u m b l i n g v e r y r a p i d l y , so t h a t t h e a n i s o t r o p i c p a r t s o f the ene r g y H a m i l t o n i a n a v e r a g e t o z e r o , and o n l y t h e i s o t r o p i c p a r t s need be c o n s i d e r e d . A c o n v e n t i o n a l s p i n H a m i l t o n i a n f o r t h i s c a s e can be w r i t t e n f o r n n u c l e i : = E l e c t r o n i c Zeeman + H y p e r f i n e + N u c l e a r Zeeman 13 3 6 - 9 e B e H 0 - I + fal (a^-l 1 - g nB nH 0-I 1) [1 ] For s i m p l i c i t y f u r t h e r d i s c u s s i o n w i l l e n t a i l o n l y t h e ca s e o f F i g 1 w i t h o n l y one n u c l e u s ( n = l ) . With a l a r g e m a g n e t i c f i e l d t h e f o l l o w i n g a p p r o x i m a t i o n s can be made: ^e e o 7 3 n n o and t h e e i g e n v a l u e s o f e q u a t i o n [ 1 ] can be o b t a i n e d ''for a s i n g l e group o f e q u i v a l e n t n u c l e i w i t h c o u p l i n g o f a.: E(ms.,mT) = g e e e H Q m s + a r t ^ - g n B n H 0 m T [3] and t h e s e a r e t h e ene r g y l e v e l s as p l o t t e d i n F i g 1. Now, i f t r a n s i t i o n s a r e i n d u c e d a c c o r d i n g t o c e r t a i n s e l e c t i o n r u l e s , one o b t a i n s f o r t h e EPR r u l e s : Am s = ±1 and Am T = 0 [4] a l l o w e d t r a n s i t i o n s a t f r e q u e n c i e s o f v ^ p R where: h v E P R = V e H o ± ™ i [5] which o c c u r around 9 GHz a t 3400 Gauss H Q i n X-Band EPR s p e c t r o s c o p y . 14 A l t e r n a t e l y , one can s o l v e e q u a t i o n [3] f o r the NMR t r a n s i t i o n s a c c o r d i n g t o t h e s e l e c t i o n r u l e s : Am s = 0 and Amj = ±1 [6] to g i v e the a l l o w e d NMR ( o r ENDOR) t r a n s i t i o n f r e q u e n c i e s f o r each group o f e q u i v a l e n t n u c l e i , t o second o r d e r a t v ^ p , where: VNMR = I" v o + a m S + a 2 / 4 9 e e e H o l v Q ± | + a 2 / 4 g E B e H Q | ( f o r p r o t o n s ) [7] where the n u c l e a r Larmor f r e q u e n c y i s g i v e n by v n = g „ 3 n H [ 8 ] o n n o For p r o t o n s , a t 3400 g a u s s , t h i s i s about 14 MHz, and i s g e n e r a l l y g r e a t e r t h a n a. T h i s means t h a t t h e l i n e s p r e d i c t e d by [7] w i l l be a l m o s t symmetri-c a l l y p o s i t i o n e d around v Q , and s e p a r a t e d by a. The a v e r a g e v a l u e o f the c e n t r e o f t h e two l i n e s i s thus s h i f t e d 2 t o a h i g h e r f r e q u e n c y f r o m v Q by a / 4 g e £ g H 0 , which does not depend on t h e l i n e under s t u d y , o n l y on a. I t i s so s m a l l t h a t i t may be i g n o r e d i n X-band f o r c o u p l i n g s l e s s t h a n 8 MHz as th e a c t u a l v a l u e o f the l i n e s c a n n o t be measured more a c c u r a t e l y t h a n 5 kHz. The app r o x i m a t e e r r o r i n the t r e a t m e n t 3 2 f o r e q u a t i o n [7] i s t h e i g n o r e d t h i r d o r d e r c o r r e c t i o n term, a / ( 9 e $ e l ~ ' 0 ) which i s 1 5 about 1 kHz o r l e s s than 1% o f the ENDOR l i n e w i d t h . T h i s t h i r d o r d e r c o r r e c t i o n has never been o b s e r v e d i n s o l u t i o n ENDOR. E q u a t i o n s [7] and [5] both p r e d i c t 2 t r a n s i t i o n s f o r t h i s c a s e and t h e s e a r e shown i n F i g 1 . I t s h o u l d be noted t h a t f o r a l l o t h e r c a s e s , eq. [ 7 ] p r e d i c t s a much s i m p l e r ENDOR spectrum. In EPR the number o f l i n e s i n c r e a s e s i n a m u l t i p l i c a t i v e s e n s e , one l i n e f o r each f n ^ m ^ l e v e l , w h i l e f o r ENDOR, t h e r e w i l l be o n l y one s e t o f l i n e s about vQ f o r each group o f e q u i v a l e n t n u c l e i . The r e l a t i v e r e s o l u t i o n o f ENDOR w i l l thus be much g r e a t e r , as t h e l i n e w i d t h s o f both t y p e s o f measurement a r e comparable. 2.2 P h e n o m e n o l o g i c a l Treatment The o b s e r v a t i o n o f t r a n s i t i o n s i s p o s s i b l e w i t h NMR o r EPR: both g i v e t h e v a l u e o f t h e h y p e r f i n e c o u p l i n g c o n s t a n t a. EPR has t h e advantage o f - g r e a t s e n s i t i v i t y , w h i l e t r u e NMR would g i v e t h e s i g n as w e l l as t h e magnitude o f a. The t e c h n i q u e o f ENDOR i s t o use t h e s e n s i -t i v i t y o f EPR t o o b s e r v e t h e NMR t r a n s i t i o n s . T h i s can be seen as w e l l from t h e f o u r l e v e l scheme o f F i g 1 . The r e l a x a t i o n t i m e s f o r t h e s e t r a n s i t i o n s a r e g i v e n by T g , ( e l e c t r o n i c ) , T , ( n u c l e a r ) , and T ( u n s p e c i f i e d c r o s s r e l a t i o n ) . Now, i f one o f the EPR t r a n s i t i o n s , say the |+ t o }- t r a n s i t i o n , (or/a'"> t o ^ t r a n s i t i o n ) i s r a d i a t e d w i t h t h e microwave f i e l d H1 w i t h enough power to e q u a l i z e t h e p o p u l a t i o n s o f t h e s e two l e v e l s , then a b a l a n c e o f r e l a x a t i o n r a t e s w i l l o c c u r . The r a t e o f a b s o r p t i o n o f microwave power from Hl w i l l e q u a l t h e l o s s by r e l a x a t i o n o v e r t h e two r o u t e s o f |a>to |a'^ and \a>tolb> t o l b ' ^ t o |a>. (See F i g 1 f o r l a b e l s on l e v e l s . ) These two pathways i n v o l v e T g and T „ . When t h i s b a l a n c e i s a c h i e v e d , the system i s i r r a d i a t e d by a s t r o n g 16 1/2 h*=g)3H 0 Cross Pathways EPR ENDOR (Elec.Zee) a MS,MT> H—>a 1/ + +> 1/ gn/3nH0-I+AI-S (NucZee) (Hyperfine) b c r e 1. Energy L e v e l d i a g r a m f o r a s y s t e m w i t h S = h and I = h showing EPR and ENDOR t r a n s i t i o n s . 17 NMR f i e l d H 2, so t h a t an a d d i t i o n a l pathway as shown i n t h e f i g u r e o f |a> t o / b > i s opened. The r e l a x a t i o n b a l a n c e w i l l a l t e r as t h i s pathway i s opened, and t h e net e f f e c t i s t h a t more en e r g y can now be abso r b e d from t h e H : f i e l d . T h i s assumes t h a t t h e pathway opened by t h e a p p l i e d NMR f r e q u e n c y i s s t r o n g enough t o s i g n i f i c a n t l y a l t e r t h e b a l a n c e . The i n c r e a s e i n t h e EPR energy a b s o r p t i o n f r o m Hx as t h i s pathway i s opened i s c a l l e d t h e ENDOR enhancement. The a d d i t i o n a l pathway argument i n d i c a t e s how one can d e t e c t t h e ENDOR enhancement. Two s e p a r a t e h i g h f r e q u e n c y s p e c t r o m e t e r s a r e -shown i n F i g 2, both a c t i n g on a sample i n :a l a r g e magnetic' f i e l d . When the s o u r c e s a r e v a r i e d i n f r e q u e n c y , o r e q u i v a l e n t l y , t h e ma g n e t i c f i e l d i s v a r i e d , a t t h e r e s o n a n t c o n d i t i o n s g i v e n by [5] ( o r [ 7 ] ) energy i s ' a b s o r b e d from t h e s o u r c e and a s i g n a l i n d i c a t i n g t h i s a b s o r p t i o n i s d e t e c -t e d . The EPR reso n a n c e c o n d i t i o n s a r e u s u a l l y o b t a i n e d by v a r y i n g t h e f i e l d H o, as t o v a r y t h e microwave f r e q u e n c y s m o o t h l y i s e x p e n s i v e and d i f f i c u l t t e c h n i c a l l y . The NMR e x p e r i m e n t a l c o n d i t i o n s a r e met i n t h e c a s e o f h i g h r e s o l u t i o n NMR by h o l d i n g t h e m a g n e t i c f i e l d c o n s t a n t and sweeping s l o w l y t h r o u g h t h e a p p r o p r i a t e r a d i o f r e q u e n c y . I f two s p e c t r o m e t e r s a r e s e t up as shown and the r e s p e c t i v e f i e l d s do not i n t e r a c t , t h e n t h e d o u b l e r e s o n a n c e e x p e r i m e n t s o f ENDOR and Dynamic N u c l e a r P o l a r i z a t i o n (DNP) can be done. In p r a c t i c e , i t i s o f t e n d i f f i c u l t t o e n s u r e t h a t t h e two f i e l d s do not i n t e r a c t . In many DNP s p e c t r o m e t e r s t h e EPR c a v i t y i s a h e l i x and i t s w i r e w a l l s s u s t a i n both microwave and r f c u r r e n t . In ENDOR, t h e NMR c o i l s o f t e n r u i n t h e q u a l i t y o f t h e microwave c a v i t y . As shown above, an a d d i t i o n a l pathway w i l l a l t e r t h e ene r g y E N D O R — D N P Microwave Source (GHz) Rf Source (MHz) Sample Detector A (NMR) Detector B (ESR) ESR and ENDOR experiment magnetic - field J NMR and DNP experiment F i g u r e 2. The ENDOR and DNP e x p e r i m e n t s : arrangements o f s o u r c e s and d e t e c t o r s . CO 19 a b s o r p t i o n b a l a n c e . I f t h e EPR a b s o r p t i o n ( d e t e c t o r B) i s m o n i t o r e d a t a c o n s t a n t f i e l d , and the r a d i o f r e q u e n c y i s swept, t h e r e ' w i l l be a change i n t h e EPR when t h e a p p r o p r i a t e f r e q u e n c y o f H 2 i s r e a c h e d . ENDOR i s thus s i m i l a r t o t h e NMR " s p i n t i c k l i n g " e x p e r i m e n t , e x c e p t t h a t t h e t i c k l i n g f r e q u e n c y i s much lower than t h e main a b s o r p t i o n f r e q u e n c y . I f t h e a p p a r a t u s i s r e v e r s e d and the NMR d e t e c t o r (A) i s m o n i t o r e d w h i l e t h e microwave s o u r c e i s swept, t h e DNP enhancements a r e see n . Both e x p e r i m e n t s y i e l d e x a c t l y t h e same i n f o r m a t i o n , and can be used i n t h e same way t o o b t a i n s i m p l i f i e d s p e c t r a . The mechanism o f both i s s i m i l a r : i t i s not s u r p r i s i n g t h a t compounds t h a t show a l a r g e DNP enhancement a l s o g i v e ENDOR. The main d i f f e r e n c e between t h e two t y p e s i s i n t h e i r a p p l i c a t i o n . DNP i s most o f t e n used t o enhance o t h e r ( n e u t r a l ) s p e c i e s i n t h e p r e s e n c e o f r a d i c a l s , and o b t a i n a b e t t e r NMR. ENDOR i s used t o s t u d y d i r e c t l y the r a d i c a l s p e c i e s . The a p p r o x i m a t e s i z e o f t h i s enhancement can be found f r o m a s u i t a b l y m o d i f i e d B l o c h - e q u a t i o n t r e a t m e n t o f the power a b s o r p t i o n . T h i s p i c t u r e i s adequate f o r most o f t h e major e f f e c t s o b s e r v e d i n t y p i c a l s o l u -t i o n ENDOR s p e c t r a and was f i r s t used by A l l e n d o e r f e r and M a k i 1 0 t o d e s c r i b e t h e ENDOR o f t h e TTBP r a d i c a l . I t i s i m p o r t a n t f o r an u n d e r s t a n d i n g o f the i n s t r u m e n t a l c o n s t r u c t i o n : t h e H 2 power n e c e s s a r y can be e s t i m a t e d from t h i s "new pathway" p i c t u r e . The B l o c h e q u a t i o n s a r e a s e t o f s i m u l t a n e o u s d i f f e r e n t i a l equa-t i o n s which d e s c r i b e t h e r e l a x a t i o n o f t h e b u l k m a g n e t i z a t i o n ' M o f ..a "sample i n terms o f T l t t h e s p i n l a t t i c e r e l a x a t i o n t i m e , T 2 , t h e s p i n - s p i n r e l a x a t i o n t i m e , Hx, t h e microwave f i e l d and y n t h e m a g n e t o g y r i c r a t i o o f t h e n u c l e u s , a b b r e v i a t e d here t o y. The s o l u t i o n o f t h e s e d i f f e r e n t i a l e q u a t i o n s , g i v e s 22 23 i n s t a n d a r d n o t a t i o n , ' the power abs o r b e d from Hi as 20 P = H u l H i-v [9] where v, the a b s o r p t i o n component, i s g i v e n from t h e B l o c h e q u a t i o n s by v - ^ [ 1 0 ] 1 + Y 2 T 2 2 ( H - H 0 ) 2 + Y 2 H i 2 T i T 2 Here H i s t h e ma g n e t i c f i e l d and H Q the c e n t e r o f t h e r e s o n a n c e . Most spec-t r o m e t e r s o p e r a t e i n a mode t h a t d i s p l a y s t h e d e r i v a t i v e o f the power absor-bed w . r . t . t h e ma g n e t i c f i e l d , so t h a t d v = g . y ^ v ^ - H ) [ 1 1 3 d H f l + ' - Y 2 V ( H - H 0 ) 2 + Y 2 H 1 2 T 1 T 2 ] 2 d e s c r i b e s t h e power d e r i v a t i v e ESR s i g n a l . Now, i f (^-) i s d i f f e r e n t i a t e d a g a i n w i t h r e s p e c t t o t h e mag n e t i c f i e l d H and t h e r e s u l t a n t ( ^ Y ^ i s s e t equal t o z e r o , t h e a l g e b r a g i v e s two symmetric v a l u e s H = H ± 7 1 + ^ H i Z T i T 2 [12] o /3~ T 2 Y f o r t h e f i e l d a t t h e d e r i v a t i v e maximum. I f t h i s f i e l d a t the maximum i s s u b s t i t u t e d i n t o t h e power s i g n a l [11] t h e a m p l i t u d e o f t h e d e r i v a t i v e a t th e maximum i s o b t a i n e d : 21 ^ X ( 2 M o Y 3 H l T 2 3 ) [ ] 3 ] d H m a x 1 6 T (1 + y ^ ^ T , ) ^ Now, the ass u m p t i o n i s made t h a t t h e ENDOR enhancement o f t h i s a m p l i t u d e i s due p r i m a r i l y t o a change i n T x , as t h e o n l y e f f e c t o f t h e a p p l i e d NMR f i e l d H 2 was to open new pathways and i n c r e a s e t h e r e l a x a t i o n r a t e ( o r s h o r t e n t h e r e l a x a t i o n time T J . I t has been shown t h a t microwave s a t u r a t i o n depends m o s t l y on a change i n T^. The change i n t h e maximum EPR s i g n a l as a f u n c t i o n o f lx i s g i v e n by t he d e r i v a t i v e o f [ 1 3 ] w i t h r e s p e c t t o T x and i s -iL_.(dy\ = _ o' 1 [ 1 4 1 d-vWmax 1 6 (1 + Y 2 H l 2 T l T 2 ) 5 ^ L and an ENDOR enhancement E can be d e f i n e d : E = A ( ^ ) = - 9 fi V ^ V . A T L [ 1 5 ] -v dH'max 5 / 1 1 6 (1 + Y 2 H I 2 T ! T 2 ) / 2 In o r d e r t o o p e r a t e a s p e c t r o m e t e r c o r r e c t l y , i t i s n e c e s s a r y t o know a t what v a l u e o f microwave f i e l d Ul t h e q u a n t i t y E has i t s maximum v a l u e . T h i s i s found by s o l v i n g ^| from [ 1 5 ] and e q u a t i n g i t t o z e r o . The a l g e b r a g i v e s Y 2 H i 2 T ! T 2 = 3/2 [ 1 6 ] 22 a t t h e maximum, which can be s u b s t i t u t e d i n t o f u r t h e r e q u a t i o n s . I f , by way o f c o m p a r i s o n the EPR power s i g n a l o f [13] i s t r e a t e d t h e same way f o r a maximum i n microwave f i e l d , by s o l v i n g ^ j — (^vj)max a n d e q u a t i n g t o z e r o , one o b t a i n s : Y 2H 1 2T 1T2 = 1/2 [17] f o r t h e maximum o f an EPR s i g n a l . T h i s means t h a t f o r t h e optimum enhance-ment s i g n a l Hi 2(END0R) = 3 H! 2(EPR) [18] o r t h a t t h e microwave power f o r maximum ENDOR enhancement i s about 5dB above t h a t power f o r t he maximum o r s a t u r a t e d EPR. Thus t h e magnetic f i e l d s h o u l d be s e t a t the EPR d e r i v a t i v e maxi-mum and t h e maximum ENDOR enhancement w i l l o c c u r a t a microwave f i e l d g r e a t e r than t h a t n e c e s s a r y f o r EPR power s a t u r a t i o n . E x p e r i m e n t a l l y t h i s i s a p p r o x i -m a t e l y c o r r e c t : b e t t e r ENDOR enhancement was o b t a i n e d i n t h i s s t u d y j u s t beyond t h e EPR s a t u r a t i o n l i m i t , r a t h e r than b e f o r e . The v a l u e o f 5dB i s n o t a good g u i d e l i n e , as t o i n c r e a s e t h e microwave power by t h i s amount u s u a l l y gave l e s s enhancement and h i g h e r n o i s e so t h a t t h e t o t a l enhancement was a l m o s t l o s t . The u s e f u l a p p r o x i m a t i o n can be c o n t i n u e d , and i f the v a l u e o f t h e maximum enhancement found by [16] i s s u b s t i t u t e d f o r unknowns i n t h e d e f i n e d enhancement o f [ 1 5 ] , a maximum a b s o l u t e ENDOR enhancement can be f o u n d : 23 ^V* ( 3 r / 2 T , 3 A T l E m a x " ~ 1 6 ( 5 / 2 ) 5 / 2 Wl{T2f [ l g ] _ 81/5 M v / U 3/ 2 ATi 1000 T h i s a b s o l u t e enhancement i s e x p e r i m e n t a l l y n o t as u s e f u l as t h e enhancement r e l a t i v e t o the EPR s i g n a l : i t c o n t a i n s t o o many unmeasurable q u a n t i t i e s . S o l v i n g f o r t h e maximum o f t h e EPR, under t h e s e ENDOR c o n d i t i o n s , by p l a c i n g t h e v a l u e f o r [16] i n t o t h e unknowns o f [ 1 3 ] , one o b t a i n s when 3 / dv\ = 9 M Q y ( T 2 ) 2 a t ( H x = ENDOR MAX) d HLx " 20 /5 ( T l ) V 2 C 2 0 ] /dy_\ W, and t h e maximum f r a c t i o n a l enhancement, F m a x i s o b t a i n e d by comparing t h i s and t h e maximum ENDOR enhancement [ 1 9 ] . Most terms c a n c e l , g i v i n g F = E /(^L) max max' MH'max = -9/10 11 [21] T h i s r e m a r k a b l y s i m p l e r e s u l t shows t h a t t o c a l c u l a t e even a r e l a t i v e enhancement, a model must be assumed where a v a l u e f o r tJi can be e v a l u a t e d . V a r i o u s models have been p r o p o s e d . S i n c e a d e t a i l e d c a l c u l a t i o n depends on t h e model (and t h u s on t h e m o l e c u l e under s t u d y ) o n l y a summary w i l l be g i v e n . 2 4 1 9 D i n s e proposed a 4 l e v e l model as i n F i g 1 w i t h t h e f o l l o w i n g a s s u m p t i o n s : 1 . a l l c r o s s r e l a x a t i o n t i m e s , T a r e v e r y l o n g ; A 2 . r e l a x a t i o n t i m e s w i t h an a p p l i e d H a r e v e r y s h o r t compared t o t h e 'T 1 t h a t e x i s t s w i t h o u t an a p p l i e d f i e l d , n With t h e s e a s sumptions D i n s e c a l c u l a t e d t h a t F t a k e s a maximum T and under t\ e [ 2 1 ] , the enhancement i s when T N - G t h e s e c o n d i t i o n s ( A ^ / T ^ i s r o u g h l y ( - 1 / 9 ) so from Fmax • ' / l O [22] o r a 1 0 % enhancement can be e x p e c t e d under o p t i m a l c o n d i t i o n s . T h i s r e s u l t , t h a t A T X / J\ = - 1 / 9 a r i s e s from an e l e c t r i c c i r c u i t a n a l o g u e o f t h e 4 l e v e l d i a g r a m and i s t o be t a k e n o n l y as a g u i d e l i n e . F r e e d has shown t h a t i n f a c t t h e maximum ENDOR a r i s e s i f T g i s much much l o n g e r t h a n T , ( n o t T g - T n ) and t h i s changes c o m p l e t e l y t h e e l e c t r i c a l c i r c u i t a n a l o g u e . The s t a t e m e n t t h a t F m a x c o u l d be 1 0 % comes from a c o r r e c t m a t h e m a t i c a l t r e a t m e n t o f t h e f o u r l e v e l s ystem, but c o n t a i n s l i t t l e v a l i d i t y when compared t o e x p e r i m e n t a l r e a l i t y . T h i s v a l u e w i l l . b e t r e a t e d . a s an;upper l.imit.to„the enhancement. A l l e n d o e r f e r and M a k i 1 0 proposed a model w i t h c r o s s r e l a x a t i o n (T ) a l l o w e d . They assumed t h a t t h e r e was e i t h e r a one s t e p o r two-step r e l a x a -2 4 t i o n . In t h e t w o - s t e p model, a l s o p r o p o s e d by F r e e d e t ' a l , t h e ENDOR opens up t h e pathway ia> t o |b> (se e F i g 1) and then t h e r e i s a two-step c r o s s r e l a x a -t i o n o f )b>to |b> ( e l e c t r o n i c ) f o l l o w e d by Jb'> t o ia'> ( n u c l e a r ) . In t h e o n e - s t e p 25 model i t i s a s i m u l t a n e o u s e l e c t r o n and n u c l e a r f l i p b t o a 1 (shown by one o f t h e T x a r r o w s ) a l o n g t h e ia> t o |b) pathway. In e i t h e r c a s e i t was assumed t h a t t h e u n s p e c i f i e d one o r tw o - s t e p r e l a x a t i o n time T u i s l e s s t h a n both T and T and t h a t T i s much l o n g e r than T . They a l s o had t o measure n e n 3 e J a c h a r a c t e r i s t i c n u c l e a r r e l a x a t i o n t ime T c f o r t h e m o l e c u l e under s t u d y (TTBP) and o b t a i n e d v a l u e * o f 1.5 x 10~ 5 s e c . They o b t a i n e d T : from EPR s a t u r a t i o n c u r v e s . The d e t a i l e d a l g e b r a o f t h e i r c a l c u l a t i o n g i v e s f o r enhancement f a c t o r s : Fmax = 0 , 3 T i / T c = , 0 9 ( o n e s t e p ) [23] Fmax = ° - 0 9 T i / T c = - 0 0 3 ( t w o s t e p ) g i v i n g a maximum enhancement o f from 0.3%to 1% o f t h e i n t e n s i t y o f t h e EPR s i g n a l , much l e s s than t h e v a l u e p r e d i c t e d by D i n s e . From t h e enhancement f a c t o r s o f [ 2 3 ] , t he e x a c t c o n d i t i o n s f o r optimum ENDOR enhancement can be more c a r e f u l l y worked o u t . A r e a s o n a b l e a s s u m p t i o n i s t h a t t h e m o l e c u l a r r o t a t i o n a l c o r r e l a t i o n v a r i e s d i r e c t l y w i t h v i s c o s i t y and i n v e r s e l y w i t h t h e t e m p e r a t u r e : T c * n/T. F u r t h e r m o r e , t h e n u c l e a r and t h e e l e c t r o n i c r e l a x a t i o n t i m e s , T. can be assumed t o v a r y w i t h t h e Larmo.r f r e q u e n c y co. and w i t h t h e above r o t a t i o n a l c o r r e l a t i o n t i m e : Now, a t h i g h t e m p e r a t u r e s , T i s s m a l l and (CD T ) 2 « 1 so t h a t i f t h e r e l a x a t i o n t i m e s o f [24] a r e used i n t h e enhancement f a c t o r e q u a t i o n s [ 2 3 ] , t h e enhancement a t h i g h t e m p e r a t u r e i s i n d e p e n d e n t o f t e m p e r a t u r e . 26 On t h e o t h e r hand, a t lower t e m p e r a t u r e s such t h a t ( u > e x c ) 2 1 but K Tc ) 2 < 1 then [24] can be used t o show t h a t t h e enhancement F v a r i e s as f o l l o w s : F = T-, ./T le t c , 0 * "eV> / ^ + " n 2 T c 2 > c c [25] e c * ^ 2 ( n / T ) 2 T h a t i s , the optimum enhancement, a l l o t h e r v a r i a b l e s b e i n g e q u a l , w i l l o c c u r a t low t e m p e r a t u r e , and i n systems o f h i g h v i s c o s i t y . These c o n d i t i o n s can be met i n s e v e r a l s o l v e n t s j u s t above t h e i r m e l t i n g p o i n t s ( t o l u e n e , n-heptane) o r by u s i n g a n o n - l o s s y s o l v e n t such as m i n e r a l o i l which has a v e r y h i g h v i s c o s i t y o v e r a l a r g e t e m p e r a t u r e range. Both t h e s e models show t h a t t h e b e s t enhancement w i l l be v e r y s m a l l . Good d e t e c t i o n and a m p l i f i c a t i o n o f t h i s s m a l l s i g n a l i s n e c e s s a r y as t h e c o n d i t i o n s may n o t always be o p t i m a l . C l e a r l y t h e enhancement w i l l depend on t h e s o l v e n t , t e m p e r a t u r e , r a d i c a l under s t u d y , d e t e c t i o n method and t h e magnitude o f the H 2 f i e l d , and t h i s d i c u s s i o n shows t h a t under t h e v e r y b e s t c o n d i t i o n s o n l y f r o m 1% t o 10% o f the EPR s i g n a l w i l l be a v a i l a b l e as ENDOR s i g n a l . For t h i s r e a s o n , l a r g e samples a r e n o r m a l l y used i n non-27 l o s s y s o l v e n t s , and t h e EPR c a v i t y must be a b l e t o c o n t a i n a l a r g e sample a t a c o n t r o l l e d t e m p e r a t u r e . T h i s w i l l l i m i t t h e number o f c h o i c e s f o r a c a v i t y d e s i g n , as t o o l a r g e a " s t a c k " h o l e n o r m a l l y c a u s e s l o s s o f t h e microwave f i e l d t h r o u g h t h e l a r g e a c c e s s s t a c k s . 2.3 The H y p e r f i n e C o u p l i n g C o n s t a n t C o r r e c t i o n o f t h e Enhancement The ENDOR i n t e n s i t y s h o u l d depend not o n l y on t h e model f o r t h e r e l a x a t i o n t i m e , but a l s o on t h e number o f p r o t o n s c o n t r i b u t i n g t o the l e v e l t h a t i s b e i n g o b s e r v e d . U s u a l l y , t h e o v e r a l l ENDOR enhancement comes from t h e r e d u c t i o n i n s p i n p o p u l a t i o n o f t h e l e v e l b e i n g s a t u r a t e d by m i c r o -wave r a d i a t i o n , i . e . t h e r f r a d i a t i o n which c o u p l e s t h e s e two l e v e l s removes many o f t h e s p i n s f r o m t h e s a t u r a t e d l e v e l t o an u n s a t u r a t e d l e v e l . However, i n a m a g n e t i c - f i e l d - m o d u l a t e d e x p e r i m e n t f o r t he c a s e o f s m a l l c o u p l i n g c o n s t a n t s , i t w i l l be v e r y d i f f i c u l t t o s a t u r a t e o n l y t h e one l e v e l : i f the t r a n s i t i o n s a r e v e r y c l o s e and both l e v e l s a r e p a r t i a l l y s a t u r a t e d b ecause o f t h e c h a n g i n g r e s o n a n c e c o n d i t i o n s , t h e ENDOR enhancement w i l l be g r e a t l y r e d u c e d . I f t h i s r e d u c t i o n w i l l c a u s e a change i n the d i f f e r e n c e i n m a g n e t i z a t i o n , M v f o r t h e two l e v e l s c o n c e r n e d , then t h e f r a c t i o n a l enhance-ment o f e q u a t i o n [21] becomes c o r r e c t e d t o AM", (a o b s e r v e d ) F = ~ T - " "Frnax [ 2 6 ] AM (a °°) z where F m a x i s as d e f i n e d i n e q u a t i o n s [21] t o [ 2 3 ] , t h e maximum enhancement f o r l a r g e c o u p l i n g s , and AM i s t h e d i f f e r e n c e i n t h e M z v a l u e f o r t h e l i n e '/Z b e i n g s a t u r a t e d and t h e M| f o r t h e l i n e c o u p l e d t o i t by t h e r f , and a i s th e o b s e r v e d s p l i t t i n g c o n s t a n t . 28 22 From t h e s o l u t i o n o f t h e B l o c h e q u a t i o n s M i s g i v e n ( i n f r e q u e n c y u n i t s ) by: 1 + V U -a))2 1 + T 22 ( u r a))2 + Y n 2 H i 2 T ! T 2 [27] Now, for.ENDOR, a shown.above i n e q u a t i o n [ 1 6 ] , t h e r e i s a microwave f i e l d r e l - a t i o n s h i o a t t h e ENDOR maximum: Y n 2 H i 2 T ! T 2 = 3/2 so t h a t , i n f r e q u e n c y u n i t s , Aco = co - coQ, the change i n M Z between t h e l e v e l o f i n t e r e s t and the next l e v e l i s : AM z(Aco Q b s) = | Mz(co = coQ) - Mz(u> f coQ) l e v e l 1. •. n e x t l e v e l n 0 j 1+372 " .1 + T 2 2(Aco) 2 + 3/2 [28] T ? 2Aco 2 w h i l e a t l a r g e c o u p l i n g s , t h e unreduced m a g n e t i z a t i o n i n t h e den o m i n a t o r o f [26] i s A M Z ( A C O -* ° ° ) = | M (co = co ) - M (co -* °°) l i m M , Aco-*00 3/5 M 0' z 2 1 + To 2(Aco) 2 0 ) 5 " 1 + TV(Aco) z + 3/2 [29] 29 T h i s w i l l c a n c e l w i t h p a r t o f [28] so t h a t t h e c o r r e c t i o n f o r " t h e Enhancement F a t any c o u p l i n g i s g i v e n from [26] by: where T 2 i s i n seconds and c o u p l i n g s a r e i n H e r t z . T h i s i s e q u i v a l e n t t o : F • T 2 V ^ 0 . 0 6 3 3 3 • F m a x ^ w i t h a i n MHz and T 2 i n m i c r o s e c o n d s . T h i s r e s u l t i s c a l l e d t h e A l l e n d o e r f e r -Maki c o r r e c t i o n f o r m u l a . Now, i n g e n e r a l F m a x i s d i r e c t l y p r o p o r t i o n a l t o t h e number o f p r o t o n s so i f t h i s i s known, t h i s c o r r e c t i o n can be used t o make e s t i m a t e s o f T 2 . G e n e r a l l y i t i s more u s e f u l t o use r e a s o n a b l e T 2 v a l u e s and check on t h e number o f p r o t o n s c o n t r i b u t i n g t o each l i n e , where each l i n e i n t e n s i t y i s c o r r e c t e d w i t h t h i s f o r m u l a . A p l o t o f t h i s c o r r e c t i o n f u n c t i o n F v e r s u s ( T 2 - a ) shows t h a t i t has a l a r g e d e c r e a s i n g e f f e c t when t h e c o u p l i n g a i s l e s s than 5 t i m e s t h e l i n e w i d t h . C o u p l i n g s g r e a t e r than 2 MHz need l i t t l e c o r r e c t i o n . The i n -h e r e n t n o i s e i n an ENDOR e x p e r i m e n t and t h e sub s e q u e n t d i f f i c u l t i e s i n m e a s u r i n g any o f t h e l i n e i n t e n s i t i e s a c c u r a t e l y , c o u p l e d w i t h t h e a p p r o x i -m a t i o n s i n [26] and unknown T 2 ' s f o r t h e v i s c o u s m i n e r a l o i l s o l u t i o n s w i l l make t h i s c o r r e c t i o n o f l i m i t e d use. The a s s i g n m e n t o f p r o t o n s i n t h e s p e c t r a o f l a r g e m o l e c u l e s i s made much-easier by a p p l i c a t i o n o f t h i s f o r m u l a . 30 The above i m p l i e s t h a t a l t h o u g h L i q u i d ENDOR has i n c r e a s e d r e s o l u t i o n o v e r EPR f o r a l m o s t i d e n t i c a l , l a r g e c o u p l i n g s (a d i f f e r e n c e o f 10 t o 20 m i l l i g a u s s i s e a s i l y v i s i b l e ) i t c a n n o t be used t o d e t e c t e x t r e m e l y s m a l l c o u p l i n g s , as t h e y w i l l have a l m o s t z e r o ENDOR enhancement. Thus ENDOR n o r m a l l y does not r e s o l v e new c o u p l i n g s t h a t a r e too s m a l l f o r EPR (a v a l u e s l e s s than 70 mG) but i s more u s e f u l f o r s e p a r a t i n g l a r g e but n e a r l y i d e n t i c a l c o u p l i n g s t h a t l e a d t o a complex m u l t i - l i n e EPR spectrum. 2.4 T h e o r e t i c a l Requirements f o r H 2 In t h i s s e c t i o n t h e d e f i n i t i o n o f inhomogeneous l i n e - b r o a d e n i n g w i l l be n e c e s s a r y . In t h e usu a l d e f i n i t i o n homogeneous EPR l i n e - b r o a d -e n i n g o c c u r s when en e r g y a b s o r b e d from H1 i s t h e r m a l l y d i s t r i b u t e d t o a l l s p i n s i n t h e t o t a l EPR spectrum. In inhomogeneous b r o a d e n i n g , H1 energy i s t r a n s f e r r e d o n l y t o s p i n s which s a t i s f y a r e s o n a n c e c o n d i t i o n i n t h e i r e n e r g y l e v e l d i f f e r e n c e s : such a group o f s p i n s i s c a l l e d a s p i n p a c k e t . A group o f s e v e r a l n o n - i d e n t i c a l s p i n - p a c k e t s i s thus c a l l e d an irihomogeneously broadened EPR l i n e . In t h i s d e f i n i t i o n , T 2 becomes t h e time f o r H x e n e r g y ' to d i f f u s e t o t h e r e s t o f s p i n p a c k e t f r o m t h e l i n e b e i n g e x c i t e d by t h e Hl f i e l d . 27 5-7 In s e v e r a l r e v i e w a r t i c l e s on l i q u i d ENDOR, Hyde ' has summarized t h e r e q u i r e m e n t s f o r ENDOR i n s o l u t i o n u s i n g t h e above d e f i n i -t i o n s . The c o n d i t i o n s f o r ENDOR a r e : - l [31] (a) (b) (c) 31 where i s the e l e c t r o n exchange t i m e , and now y e must be d i s t i n g u i s h e d from S Y N • In e q u a t i o n [31] t h e f i r s t i n e q u a l i t y , (a) s t a t e s t h a t t h e EPR l i n e must be inhomogeneously broadened. The en e r g y e x t r a c t e d f r o m H x must d i f f u s e t h r o u g h t h e s p i n p a c k e t more s l o w l y than i t does t o the l a t t i c e and hence t o o t h e r r a d i c a l s . Depending on t h e model f o r ENDOR enhancement used (see s e c t i o n 2.3) and the t y p e o f e l e c t r o n exchange i n t h e syste m , one can s u b s t i t u t e T ( c r o s s r e l a x a t i o n ) o r T f o r s p i n d i f f u s i o n . A U I n e q u a l i t y (b) s t a t e s t h a t t h e system must be u n d e r g o i n g m i c r o -wave power s a t u r a t i o n ( i e . Y e 2 ' H i 2 T 1 T 2 > 1) from t h e s o l u t i o n o f t h e B l o c h 22 J ' e q u a t i o n s . In t h i s i n t e r p r e t a t i o n ( T ^ ) 2 i s an avera g e time f o r r e c o v e r y o f t h e system t o thermal e q u i l i b r i u m , and ( Y O H J ) " 1 i s an approx-imate time f o r an e l e c t r o n i c o r Am<. t r a n s i t i o n t o o c c u r . S i n c e (yrHz)~1 i s a time f o r a n u c l e a r t r a n s i t i o n Anij t o o c c u r , e q u a l i t y (c) i s m e r e l y s t a t i n g t h a t t h e s e two times must be comparable f o r H 2 t o a l t e r t h e p o p u l a t i o n s a t a l l : i f t h e r e i s t o be any change i n the s a t u r a t i o n o f t h e l e v e l s , t h e two p r o c e s s e s o f n u c l e a r and e l e c t r o n i c t r a n s i -t i o n s must~be c o m p e t i t i v e . For f r e e r a d i c a l s i n s o l u t i o n , T x and T 2 a r e about 1 0 " 5 t o 1 0 ~ 7 s e c ; i n t h e l a r g e r T£Q1.3 mode c a v i t i e s t h e microwave f i e l d Hi i s about 30 mi H i Gauss a t 25 mw. S i n c e Y E / Y N = 658, then H 2 = ( y E / Y N ) X Hi = 20 Gauss i n t h e r o t a t i n g frame. To g e n e r a t e from 10 to 100 Gauss i n a l a r g e two-t u r n c o i l , 20 amps o r more a r e needed o r 100 t o 1000 Watts o f r f power, as 27 shown by Hyde. Such c o n d i t i o n s a r e met on t h e c o m m e r c i a l l y a v a i l a b l e 19 V a r i a n s p e c t r o m e t e r . U s i n g t h e upper l i m i t model o f D i n s e where t h e r e i s no c r o s s r e l a x a t i o n , T = 0, and t h e n o t a t i o n T f o r e l e c t r o n i c r e l a x a t i o n , A C 32 f o r n u c l e a r r e l a x a t i o n , t he B l o c h e q u a t i o n s g i v e WV 2 n " 1 a " d V ^ W i * * 1 I K ] f o r microwave s a t u r a t i o n and c o m p e t i t i v e nuclear, s a t u r a t i o n . But by [22] i t was assumed from e l e c t r i c a l c i r c u i t a n a l o g u e s t h a t F was a maximum when T n - T e and o n l y a L c h a n g e i n T x was r e s p o n s i b l e f o r F, so t h a t t h i s model has o n l y one r e q u i r e m e n t - f o r t h e ENDOR r f mag n e t i c f i e l d . H 2 > ^ H L [33] Y n 2n T h i s g i v e s about t h e same e s t i m a t i o n , t h a t t he s t r e n g t h o f H 2 must be around 100 Gauss i n t h e r o t a t i n g frame o f the B l o c h e q u a t i o n s o l u t i o n , d e p e n d i n g on t h e v a l u e s s e l e c t e d f o r T and T . ^ 3 2e 2n 2.5 D e t e c t i o n Methods The s m a l l s i z e o f t h e ENDOR s i g n a l ( 1 0 ~ 9 V o l t ) o r a b o u t 1% o f ESR s i g n a l s s u g g e s t s t h a t a t l e a s t a d o u b l e - m o d u l a t i o n - s c h e m e - w i l T be r e q u i r e d . I f t h e d e t e c t i o n c i r c u i t s a r e d i s c r i m i n a t i n g enough t h i s d o u b l e c o d i n g and d e c o d i n g s h o u l d be s u f f i c i e n t f o r an a d e q u a t e - s i g n a l - t o - n o . i s e r a t i o . The c h o i c e o f m o d u l a t i o n f r e q u e n c i e s i s l i m i t e d f o r c e r t a i n t e c h n i c a l r e a s o n s . For optimum S/N, a t l e a s t one o f t h e L o c k - i n a m p l i f i e r s s h o u l d be s e t t o a h i g h f r e q u e n c y , above 10 kHz, but w e l l s e p a r a t e d from t h e A.F.C. c i r c u i t f r e q u e n c y . Then, i n o r d e r t o have a s i g n a l p a s s e d by t h e f i r s t s t a g e , t h e second f r e q u e n c y s h o u l d be much l o w e r . 33 One can d e c i d e which f r e q u e n c y t o use by a c o n s i d e r a t i o n o f the s i z e o f o b s e r v e d c o u p l i n g s . The t r a n s i t i o n whose p o p u l a t i o n d i f f e r e n c e i s b e i n g changed by a p p l i e d r f must r e a c h thermal e q u i l i b r i u m w i t h t h e l a t t i c e b e f o r e t h e next t r a n s i t i o n t o which i t i s c o u p l e d i s s a t u r a t e d by t h e m o d u l a t i o n o f t h e m a g n e t i c f i e l d . T h i s means t h a t t h e m o d u l a t i o n dH d t v e l o c i t y -rr must have an upper l i m i t o f aloauss) [ 3 4 ] Ti (sec) L J dH a ( g a u  d t The m o d u l a t i o n f r e q u e n c y w i l l be r e l a t e d t o t h e depth o f magnetic f i e l d m o d u l a t i o n A H q and t h i s v e l o c i t y by: - d H / AW vmod - d t / A H o <: a -1 T i A H Q s e c [35] The s m a l l e s t c o u p l i n g measured i s about .02 Gauss, the s p e c t r a a r e around 30 Gauss wide, but t h e depth o f m o d u l a t i o n A H Q i s l i m i t e d by t h e ENDOR c a v i t y t o be about 10 Gauss. E x p e r i m e n t a l e l e c t r o n i c J1's range from 1 0 - l t t o 1 0 " 6 s e c . Thus l i m i t s f o r t h e f r e q u e n c y a r e f o u n d by s u b s t i t u t i o n t o be: v d < 2000 Hz ( s h o r t and [36] vmod < 2 0 H z (- 1 o n s T i } 34 Thus, t o c o v e r t h e e n t i r e range o f e x p e c t e d r a d i c a l s , t h i s f r e q u e n c y s h o u l d be as low as p o s s i b l e and f o r t h i s s p e c t r o m e t e r t h e f i e l d m o d u l a t i o n f r e q u e n c y l i m i t o f : was c h o s e n . The e x a c t f r e q u e n c y was d e t e r m i n e d by s e a r c h i n g f o r minimum n o i s e and maximum s e n s i t i v i t y i n the l o c k - i n a m p l i f i e r i n use: The r f i s then f r e q u e n c y - m o d u l a t e d a t t h e h i g h e r f r e q u e n c y , 10 kHz, and because ENDOR l i n e w i d t h s a r e about 0.1 MHz t h i s m o d u l a t i o n has l i t t l e e f f e c t on t h e l i n e s h a p e and one can go to h i g h e r f r e q u e n c i e s 1 0 w i t h o u t d i s t o r t i o n t o e l i m i n a t e i n t e r f e r e n c e w i t h o t h e r systems such as th e A.F.C. c i r c u i t s . A l l e n d o e r f e r and M a k i 1 0 mention two i m p o r t a n t a s p e c t s o f the Zeeman f i e l d m o d u l a t i o n . U s i n g e q u a t i o n [21] and a F o u r i e r s e r i e s t r e a t m e n t o f t h e r e l a x a t i o n o f a s p i n p a c k e t on and o f f t h e ENDOR r e s o n a n c e t h e y c a l c u l a t e t h a t t h e ENDOR i n t e n s i t y I / I Q a t 1>ong time ( e q u i l i b r i u m ) t o be dH I / I o = n£ l S(n).[l M j l f f l , ) * ] - 1 [ 3 7 ] z S(n) l where S(n) = — s i n {-J^—) i s t h e nth component o f t h e F o u r i e r s e r i e s , and n 113 I i s the i n t e n s i t y w i t h no m o d u l a t i o n . The f i r s t p o i n t i s t h a t e q u a t i o n [37] p r e d i c t s a maximum i n t h e ENDOR i n t e n s i t y when ;the m o d u l a t i o n v e l o c i t y dH d t i s s m a l l o r z e r o . A l l e n d o e r f e r and Maki f i n d t h i s t o be t r u e both 35 t h e o r e t i c a l l y and e x p e r i m e n t a l l y . A t h e o r e t i c a l e x p a n s i o n o f [37] w i t h n = 200 can be made t o f i t t h e i r i n t e n s i t i e s measured a t v a r i o u s f r e q u e n c i e s . The h i g h e s t i n t e n s i t y i s o b t a i n e d a t l o w e s t f r e q u e n c i e s . S e c o n d l y , t h e g e n e r a l s o l u t i o n f o r t h e maximum ENDOR p r e d i c t s a phase s h i f t AG f o r t h e ENDOR maximum o f AG • t a n - 1 ( n ^ $ T J [38] i n t h e phase a n g l e o f the Zeeman f i e l d m o d u l a t i o n w i t h r e s p e c t t o the phase a t t h e maximum EPR. I t i s d i f f i c u l t t o p r e d i c t t h e s i z e o f A0 from [38] as i t i s d i f f e r e n t f o r each component n o f t h e s o l u t i o n o f t h e F o u r i e r s e r i e s . A l l e n d o e r f e r and M a k i 1 ^ o b s e r v e d t h i s s h i f t i n a q u a l i t a t i v e manner. I t was a l s o o b s e r v e d i n t h i s s t u d y , sometimes r u n n i n g as h i g h as 12 5 ° , d e p e n d i n g on t h e m o d u l a t i o n f r e q u e n c y . These o b s e r v a t i o n s mean t h a t one must modulate t h e Zeeman f i e l d a t as low a f r e q u e n c y as p o s s i b l e , and e x p e c t some l a r g e phase s h i f t when t h e a p p a r a t u s i s s w i t c h e d t o ENDOR d e t e c t i o n . Phase: s h i f t s n e a r 90° mean t h a t no enhancement would be v i s i b l e , even i f the r e s t o f the a p p a r a t u s was o p t i m i z e d , u n l e s s t h e f i e l d m o d u l a t i o n a m p l i f i e r were s h i f t e d .in phase. 2.6 S p e c t r o m e t e r Requirements These l a s t s e c t i o n s s e t s e v e r a l l i m i t s and r e q u i r e m e n t s on. t h e s p e c t r o m e t e r . I t i s d i f f i c u l t t o p r e d i c t which o f the c o n d i t i o n s w i l l be h a r d e s t t o - o b t a i n i n a p a r t i c u l a r . s a m p l e . Some o f t h e s e r e q u i r e m e n t s can be summarized i n t h e f o l l o w i n g s e c t i o n . 36 Rf f i e l d s from 10 t o 100 Gauss can be o b t a i n e d i n two ways: low power r f a m p l i f i e r s and m u l t i - - t u r n c o i l s , o r l a r g e powers. E x p e r i m e n t s w i t h low power were not v e r y f r u i t f u l i n t h i s s t u d y . Even w i t h l a r g e power a m p l i f i e r s i n t h e 50 t o 500 Watt r a n g e , t h e l a r g e c a v i t y r e q u i r e d by t h e l a r g e samples s e t s c e r t a i n l i m i t s . To o b t a i n f r e q u e n c i e s i n t h e ENDOR range o f 10 MHz on e i t h e r s i d e o f 15 MHz ( f r e e p r o t o n ) and t o o b t a i n r f f i e l d s anywhere near 10 Gauss i n t h e r o t a t i n g frame, good impedance m a t c h i n g between t h e a m p l i f i e r i n use (commonly 50 a o u t p u t impedance) and t h e c o i l must be o b t a i n e d . In a l a r g e two o r f o u r l o o p c o i l t h e i n d u c t a n c e was f o u n d t o be about 0.1 t o 0.3 yH. Any s e l e c t e d r e s i s t a n c e i n s e r i e s w i t h t h e c o i l c r e a t e s good m a t c h i n g and l e s s r e f l e c t i v e i n t e r f e r e n c e . T h i s t e c h n i q u e has been used s e v e r a l t i m e s f o r s o l i d ENDOR s t u d i e s . A drawback f o r l i q u i d s i s t h a t most o f t h e r f power i s c o n v e r t e d i n t o heat i n t h e r e s i s t o r , and not i n t o h i g h m a g n e t i c f i e l d s . Two d e v i c e s were used i n t h i s s t u d y t o o b t a i n m a t c h i n g , and o n l y t h e g e n e r a l methods w i l l be d e s c r i b e d h e r e . A p a r a l l e l tank c i r c u i t o r LC c i r c u i t can be d e v i s e d t o g i v e good m a t c h i n g i n t h e d e s i r e d f r e q u e n c y range. For t h e range o f 5 t o 30 MHz a c a p a c i t o r w i t h a v a r i a b l e t u n i n g range o f 25 to 2500 p f was s e l e c t e d . The Q o f such a c i r c u i t i s v e r y low, around 10 t o 20. A d d i t i o n a l m a t c h i n g was o b t a i n e d w i t h r f s t e p - u p t r a n s f o r m e r s which i n -c r e a s e d t h e c u r r e n t i n the tank c i r c u i t . The c u r r e n t w i t h such d e v i c e s i s d i f -f i c u l t t o c a l c u l a t e . I f i t i s assumed t h a t t h e r e s i s t a n c e ( e f f e c t i v e ) o f the tank c i r c u i t i s near one ohm, some p r e r e q u i s i t e s f o r s p e c t r o m e t e r c o n s t r u c t i o n can be'found:.,- T h i s r e s i s t a n c e w i l l depend on t h e Q f a c t o r , s k i n d e p t h , c o i l c o n f i g u r a t i o n , and f r e q u e n c y o f measurement, b u t i n a l l c a s e s , s h o u l d be v e r y low. Most o f t h e power i n t h e a m p l i f i e r s , ( a ) , w i l l now be d i s s i p a t e d 37 2 i n r e s i s t i v e l o s s e s i n t h e tank c i r c u i t ( T C ) : P = i , 2 R = i^„R T , and i f a a i L i L 300 w a t t s a r e i n use ( t y p i c a l f o r t h i s s t u d y ) t h e n 17 amps o f c u r r e n t a t l e a s t w i l l f l o w i n t h e tank c i r c u i t b e f o r e t r a n s f o r m e r s a r e added. T h i s l a r g e c u r r e n t p l a c e s s e v e r a l r e s t r i c t i o n s on c a v i t y c o n s t r u c t i o n : t h e tank c i r c u i t must be c e r t a i n l y c o o l e d , and c a v i t y s t a b i l i t y w i l l be d e c r e a s e d un-l e s s t h i s i s done c a r e f u l l y . Samples must be p r e p a r e d i n such a way t h a t T x and AT J can be o p t i m i z e d . T h i s means t h a t s o l v e n t t e m p e r a t u r e and t h u s v i s c o s i t y w i l l be v a r i e d , and a good t e m p e r a t u r e c o n t r o l o f t h e sample w i l l be n e c e s s a r y . In i 9 g e n e r a l t h e sample w i l l be l a r g e , v i s c o u s and v e r y c o n c e n t r a t e d (10" M). I t must be p o s s i b l e t o outgas such samples c o m p l e t e l y , and c o n t r o l t h e r a d i c a l c o n c e n -t r a t i o n . I t must a l s o be p o s s i b l e t o dewar t h e s e l a r g e samples p e r f e c t l y , e s p e c i a l l y a g a i n s t t h e l a r g e h e a t i n g e f f e c t s c a u s e d by t h e h i g h r f f i e l d t h a t s u r r o u n d s i t . The microwave c a v i t y must be c a p a b l e o f s u s t a i n i n g an H1 f i e l d l a r g e enough t o microwave s a t u r a t e t h e s p i n p a c k e t s , and must a l s o s u s t a i n an H 2 f i e l d o f around 10 Gauss i n t h e r o t a t i n g frame i n ; o r d e r t o i n i t i a t e n u c l e a r r e l a x a t i o n pathways. The r a d i o f r e q u e n c y ( r f ) f i e l d H 2 must be o f c o n s t a n t a m p l i t u d e . The tank c i r c u i t must be t u n a b l e o v e r t h e range o f c o u p l i n g s e x p e c t e d . High s e n s i t i v i t y f o r t h e v e r y weak (1%) enhance-ments can be o b t a i n e d w i t h l a r g e samples and a d o u b l e m o d u l a t i o n scheme: h i g h f r e q u e n c y m o d u l a t i o n (fm) f o r t h e r f f i e l d and low f r e q u e n c y f o r the ma g n e t i c f i e l d m o d u l a t i o n . I t i s a l s o p o s s i b l e t o modulate t h e microwave f i e l d Hi, t o o b t a i n t h i s second m o d u l a t i o n , b ut t h i s would n o t a l l o w t h e a p p a r a t u s t o be s i m p l y an a d d i t i o n t o an e x i s t i n g s p e c t r o m e t e r , as Hi i s a l r e a d y modulated by t h e A.F.C. c i r c u i t i n most EPR s p e c t r o m e t e r s . F i n a l l y , t h e a p p a r a t u s must have h i g h thermal s t a b i l i t y , as t h e EPR r e s o n a n c e w i l l be m o n i t o r e d as a f u n c t i o n o f t h e H 2 f r e q u e n c y t o 38 p l o t t h e ENDOR enhancement. The EPR r e s o n a n c e c o n d i t i o n must n o t change dur-i n g an ENDOR s c a n , and t h i s means t h e microwave f i e l d Hi and t h e magnetic f i e l d H Q must s t a y v e r y c o n s t a n t d u r i n g a s c a n . T h i s problem i s c o m p l i c a t e d by th e f a c t t h a t H 2 r f f i e l d s as l a r g e as d e s i r e d w i l l c a u s e h e a t i n g o f many components i n t h e microwave c a v i t y , and can a l s o c a u s e d i n s t a b i l i t y i n s e v e r a l s e n s i t i v e e l e c t r o n i c c i r c u i t components. S p e c i a l s h i e l d i n g and f i l t e r i n g o f s e l e c t e d components may be n e c e s s a r y t o p r o t e c t s e n s i t i v e c i r c u i t s and e n s u r e l o n g term s t a b i l i t y . 2.7 Tre a t m e n t o f Data I t i s seldom s u f f i c i e n t t o measure o n l y t h e ENDOR s p e c t r a i n s o l u t i o n . A l t h o u g h t h e a c c u r a c y o f t h e measured c o u p l i n g s i s much b e t t e r from ENDOR than EPR, the a s s i g n m e n t o f v a r i o u s c o u p l i n g s t o m o l e c u l a r p o s i t i o n s i s seldom s i m p l e as t h e r e i s l i t t l e s p e c t r a l i n f o r m a t i o n on the number o f n u c l e i . A p p l i c a t i o n o f t h e T 2 c o r r e c t i o n f o r m u l a c l a r i f i e s some o f the a s s i g n m e n t s , b u t t h e as s i g n m e n t s a r e n o t as c l e a r as can be o b t a i n e d from EPR. In each c a s e s t u d i e d , t h e f i r s t guess from t h e ENDOR ass i g n m e n t was used t o s i m u l a t e an EPR spectrum. T h i s was compared w i t h a w e l l r e s o l v e d EpR s p e c t r u m o b t a i n e d i n a t h i n n e r s o l v e n t . When an ass i g n m e n t gave a good f i t t o both t h e EPR and t h e ENDOR, i t was c o n s i d e r e d c o r r e c t . Then, u s i n g t h e c o r r e c t i o n f o r m u l a [ 2 8 ] and t h e number o f p r o t o n s f r o m t h e a s s i g n m e n t , an e s t i m a t e o f T 2 was o b t a i n e d from an a c c u r a t e s i m u l a t i o n o f t h e ENDOR spectrum. T h ese ENDOR s i m u l a t i o n s w i l l not be i n c l u d e d as i n g e n e r a l t h e y l o o k i d e n t i c a l t o t h e measured s p e c t r a e x c e p t f o r b a s e l i n e d r i f t , and goodness o f f i t c a n n o t be based on t h e s e s i m u l a t i o n s a l o n e . 39 A l l o f t h e m o l e c u l e s under c o n s i d e r a t i o n had s p e c t r a w i t h l a r g e numbers o f EPR l i n e s . An i m p o r t a n t p a r t o f t h e t h e o r e t i c a l r e q u i r e m e n t s a r e t h e programs f o r o b t a i n i n g a c c u r a t e s i m u l a t i o n s . 27 The program used i n t h e e a r l i e r p a r t o f t h i s s t u d y was ESRPLOT, a program f o r EPR s i m u l a t i o n used i n t h i s department. I t g e n e r a t e s a s t i c k d i agram from a m o d i f i e d form o f e q u a t i o n [5] w i t h i n t e n s i t i e s as s u p p l i e d by t h e d a t a . Then a L o r e n t z i a n o r G a u s s i a n a b s o r p t i o n o r d e r i v a t i v e c u r v e w i t h p r o p e r l i n e w i d t h i s c a l c u l a t e d and f i t t e d around each o f t h e above l i n e p o s i t i o n s . The s p e c t r u m i s s c a l e d and p l o t t e d w i t h t h e IBM 360/67 computer on a Calcomp p l o t t e r . These s p e c t r a can then be compared d i r e c t l y w i t h t h e measured s p e c t r a . T h i s p r o c e d u r e worked w e l l f o r a few o f t h e m o l e c u l e s s t u d i e d , but once t h e number o f l i n e s i n c r e a s e d beyond 1000, the c o s t o f f i t t i n g a L o r e n t z i a n e n v e l o p e on each l i n e becomes r e m a r k a b l y h i g h . A l t h o u g h the program was o p t i m i z e d s e v e r a l t i m e s d u r i n g t h e c o u r s e o f t h i s s t u d y , t h e h i g h i n e f f i c i e n c y o f t h i s t y p e o f f i t t i n g on a 360/67 produces v e r y e x p e n s i v e s i m u l a t i o n s . Some s i m u l a t i o n s r e q u i r e d o v e r 5 x 1 0 8 e x p o n e n t i a l s t o be c a l c u l a t e d . When t h e number of l i n e s r o s e above 100,000 as i n t h e c a s e o f DPPH, s p e c i a l p r e c o n s t r u c t i o n t e c h n i q u e s were employed. In s p i t e o f t h e s e d i f f i c u l t i e s most o f t h i s s t u d y was done w i t h t h i s program. The major advantage o f ESRPLOT i s t h a t v a r i o u s i n t e n s i t i e s can be used f o r d i f f e r e n t l i n e s i n a g roup, and thus i t can s i m u l a t e q u i t e w e l l s p e c t r a w i t h l i n e w i d t h v a r i a t i o n . Near t h e c o m p l e t i o n o f t h i s work, a l i s t i n g o f SESRS ( S i m u l a t e d ESR S p e c t r a ) was r e c e i v e d f r o m Dr. A l l e n d o e r f e r , SUNYAB. T h i s program c a l c u l a t e s once a s i n g l e L o r e n t z i a n d e r i v a t i v e l i n e which i s s t o r e d and 40 a p p l i e d t o each l i n e i n a group. The completed group e n v e l o p e i s s t o r e d and a p p l i e d t o t h e next l a r g e r l i n e group: t he spectrum i s b u i l t up from t h e c e n t r e o f an e n v e l o p e t o the o u t s i d e . The completed s p e c t r u m i s s c a l e d and p l o t t e d as above. SESRS was m o d i f i e d by t h i s a u t h o r and i s now a v a i l a b l e under t h e name QUICK. M o d i f i c a t i o n s i n c l u d e d t he removal o f e x t r a n e o u s s c a l i n g r o u t i n e s and t h e f o l d i n g o f each L o r e n t z i a n l i n e and o f each group e n v e l o p e about t h e c e n t r e so t h a t o n l y one h a l f o f each need be c a l c u l a t e d . By comparison 20 e x p o n e n t i a l s and 1 0 5 s i m p l e m u l t i p l i c a t i o n s a r e needed f o r a t y p i c a l s p e c t r u m . The program can a l s o s i m u l a t e ENDOR s p e c t r a as a f u n c t i o n o f T 2 d i r e c t l y f r o m d a t a f o r t h e EPR s i m u l a t i o n . T h i s second m o d i f i c a t i o n was used f o r c a l c u l a t i o n o f T 2 wherever i t i s l i s t e d i n t h i s s t u d y . The main d i s a d v a n t a g e ' ^ o f QUICK i s t h a t a l l l i n e i n t e n s i t i e s a r e f i x e d , and o n l y n u c e l i o f I = h and I = 1 can be used a t p r e s e n t , w i t h no more than 3 s e t s i n each group o f e q u i v a l e n t n u c l e i . With t h e s i m u l a t i o n o f EPR and ENDOR t h e problem o f a s s i g n m e n t o f c o u p l i n g s when t h e r e a r e a l a r g e number o f p r o t o n s becomes s i m p l i f i e d . The s t u d y o f s o l u t i o n ENDOR c a n n o t be complete w i t h o u t p r o p e r s i m u l a t i o n o f t h e EPR, as s e v e r a l assignments o f n u c l e i can g i v e i d e n t i c a l ENDOR, and o n l y t h e EPR s i m u l a t i o n can be used f o r c h e c k i n g t h e c o r r e c t a s s i g n m e n t . 2.8 S c a t c h a r d T h e o r y In t h e s t u d y o f t h e TTBP hydrogen bond complex f o r m a t i o n , i t was n e c e s s a r y t o t r e a t t h e c o u p l i n g s o b t a i n e d by ENDOR w i t h a s p e c i a l p l o t t o 28 e n s u r e t h a t t r u e complex f o r m a t i o n was b e i n g o b s e r v e d . D e r a n l e a u has 41 p o i n t e d o u t t h a t o n l y t h e S c a t c h a r d p l o t w i l l e n s u r e t h a t t h e t r e a t m e n t o f t h e d a t a i s f a i r l y v i s u a l i z e d . F o r the sake o f c o m p l e t e n e s s , t h e t h e o r y as i t a p p l i e s t o EPR and ENDOR couDlings i n s o l u t i o n i s i n c l u d e d h e r e . T h i s s e c t i o n i s a b r i e f o u t l i n e o f a p r o c e d u r e f o r o b t a i n i n g a f o r m a t i o n c o n s t a n t o f a weak, pa r a m a g n e t i c complex by means o f c o u p l i n g s o b t a i n e d from ENDOR. I t i s a g e n e r a l i z a t i o n o f t h e p r o c e d u r e o f D e r a n l e a u 29 and P e r s o n f o r NMR c h e m i c a l s h i f t s t u d i e s . Recent work i n t h i s d e p a r t -30 ment used a s i m i l a r t e c h n i q u e f o r l i g a n d exchange as s t u d i e d by EPR. The t r e a t m e n t i s n o t s p e c i f i c t o hydrogen bondings but can be used f o r a s t u d y o f any complex t h a t i s formed i n s u f f i c i e n t c o n c e n t r a t i o n t o a f f e c t ENDOR c o u p l i n g s . Assume t h a t a r a d i c a l , R, o r i g i n a l l y i n an i n e r t s o l v e n t i s u n d e r g o i n g a 1:1 complex f o r m a t i o n w i t h some p r o t o n d o n a t i n g s o l v e n t , HS, t o form t he hydrogen bonded s p e c i e s R---H—S. Some e q u i l i b r i u m w i l l be e s t a b l i s h e d between R and RHS as f o l l o w s R + H-S N " R-H-5 [39] where.K i s the f o r m a t i o n c o n s t a n t . A s a t u r a t i o n f r a c t i o n f o r t h i s e q u i l i b r i u m i s d e f i n e d by S, which t a k e s t he v a l u e o f z e r o when t h e r e i s no complex and has a d e f i n e d v a l u e o f one when a l l o f the r a d i c a l i s t i e d up i n the complex. The measured v a r -i a b l e s w i t h S = 0 can be found i n the i n e r t s o l v e n t b u t any v a l u e s f o r S = 1 ca n n o t be found u n l e s s t h e complex R-HS.-can be i s o l a t e d and p u r i f i e d . F o r 42 r a d i c a l s t h i s complex i s u s u a l l y u n o b t a i n a b l e . These r e s t r i c t i o n s f o r S can be o b t a i n e d by d e f i n i n g S = [RHS]/.[R-] where [ R 0 ] - [R] = [RHS], and [ H S Q ] - [HS] = [RHS] [40] a r e t h e i n i t i a l c o n c e n t r a t i o n s . Now, t h e e q u i l i b r i u m c o n s t a n t becomes !RHS] _ [RHS] [41] >J L H S J { [ R J - [ R H S ] H [ H S l-[RHSJ} K = N o r m a l l y , [ R Q ] i s ke p t s m a l l i n EPR to a v o i d s p i n - s p i n i n t e r a c t i o n s , so t h a t t h e r e i s much l e s s r a d i c a l than s o l v e n t and thus l e s s complex as w e l l [ H S Q ] » [R] [42] » [RHS] From [41] t h e f o r m a t i o n c o n s t a n t becomes K - . [RHS] [43] K " {[R 01-[RHS]} [ H S 0 J I f t h e r e i s r a p i d exchange t h e aver a g e c o u p l i n g o b s e r v e d , a, w i l l be a w e i g h t e d a v e r a g e 43 a " P R a R + PRHS aRHS t 4 4 ] where t h e P^'s a r e the f r a c t i o n s o f the r a d i c a l i n t h e 2 s t a t e s , f r e e i n an i n e r t s o l v e n t , and t o t a l l y bonded t o HS. The a v a l u e s a r e a ^ f o r t h e pure s o l v e n t and a^<. f o r t h e u n o b t a i n a b l e complex. Now, by d e f i n i t i o n o f P^ and S, t h e s e f r a c t i o n s can be f o u n d a s : P R = 1 - s i - [P.HS]/[R 0 ] - jrrm^- [45] and P = 1 - P = KE H£o] - T461 RHS 1 R 1+K[HS J L J Now, t h e measured e f f e c t 6 i s the d i f f e r e n c e between t h e c o u p l i n g measured i n i n e r t s o l v e n t , a^, and the a v e r a g e c o u p l i n g o b s e r v e d when complex f o r m a t i o n t a k e s p l a c e : 6 = a - a R [47] D e f i n i n g 6 Q = ( a ^ - a '^), as t h e maximum measured e f f e c t o b s e r v a b l e when S = 1, then 44 which g i v e s f o r [ 4 6 ] : 6 = PRHS V 6 = K [ H S 0 ] ( 6 Q - 6) [48] f o r t h e measured e f f e c t . T h i s can be r e a r r a n g e d t o g i v e : T H y = - K 6 ' + K 6 o [ 4 9 ] 31 which i s the S c a t c h a r d p l o t . A l i n e a r p l o t o f 6/[HS ] vs 6 w i l l c o n f i r m t h e 1:1 b i n d i n g p r o c e s s , t h e s l o p e i s K and t h e i n t e r c e p t g i v e s 6 . 28 D e r a n l e a u shows t h a t i t i s i m p o r t a n t t o keep t h e e q u i l i b r i u m s a t u r a t i o n f a c t o r S i n t h e range 0.2 < S < 0.8. S t a t i s t i c a l t r e a t m e n t o f the e r r o r s i n o b s e r v a t i o n s has shown t h a t p o i n t s o u t s i d e t h i s range a r e s u s c e p t i b l e t o v e r y l a r g e e r r o r s . T h i s means t h e o b s e r v e d change <5 must l i e 0.2 6 Q < 6 < 0.8 6Q. N o n - l i n e a r i t y i n t h i s p l o t w i l l i n d i c a t e t h a t t h e i n i t i a l a s s u mption o f 1:1 complex f o r m a t i o n i s i n c o r r e c t , a l t h o u g h on c h e m i c a l g r o u n d s , t h i s i s v e r y hard t o a c c e p t f o r a hydrogen-bonded complex. A l i n e a r S c a t c h a r d p l o t w i l l thus c o n f i r m t h e ass u m p t i o n o f a 1:1 complex f o r m a t i o n , and g i v e t h e K v a l u e and e x p e r i m e n t a l l y u n o b t a i n a b l e 6 v a l u e a l l w i t h a s i n g l e t r e a t m e n t o f t h e d a t a . The sn thus o b t a i n e d can o o be used t o e l i m i n a t e p o i n t s o u t s i d e D e r a n l e a u ' s r a n g e , even i f t h e pure complex RHS c a n n o t be i s o l a t e d and measured. T h i s makes i t a much more u s e f u l t r e a t m e n t than any o f t h e o t h e r r e c i p r o c a l p l o t s commonly used t o t r e a t NMR 3? data. 45 CHAPTER THREE EXPERIMENTAL I n t r o d u c t i o n 33 34 Some p r e v i o u s work ' i n d i c a t e s t h a t q u i t e low r a d i o f r e q u e n c y ( r f ) power might be s u f f i c i e n t t o i n d u c e ENDOR enhancements, i f t h e c o i l s s u p p l y i n g t h e NMR H 2 f i e l d a r e q u i t e c l o s e t o t h e sample. The i n i t i a l s e a r c h f o r ENDOR was c a r r i e d o u t u s i n g a 25 Watt ENI r f a m p l i f i e r , w i t h t h e s o l i d ENDOR s p e c t r o m e t e r - e l e c t r o n i c s c o n s t r u c t e d 2 i n t h i s d e partment. A V a r i a n T E 1 0 2 c a v i t y was used w i t h many d i f f e r e n t r f c o i l c o n f i g u r a t i o n s as shown i n F i g 3. Most o f t h e s e c o i l s were p l a c e d i n s i d e t h e t e m p e r a t u r e dewar, q u i t e c l o s e t o t h e sample, and u t i l i z e d t h e tank c i r c u i t shown i n F i g 3 f o r m a t c h i n g t he a m p l i f i e r t o t h e r f c o i l . No ENDOR e n h a n c e m e n t . s i g n a l s w e r e i o b s e r v e d • w i t h any o f t h e s e c o i l c o n f i g u r a t i o n s . In some c a s e s , t h e l a r g e gauge w i r e Figure 3. C o i l s used f o r low power attempts and assoc i a t ed tank c i r c u i t s . 47 would n o t pass enough c u r r e n t t o g e n e r a t e more th a n 1 t o 2 Gauss f o r H 2. I f a s m a l l e r gauge w i r e was employed 4 Gauss c o u l d be o b t a i n e d but t h e s l i g h t e s t motion o f t h e s e c o i l s from r f h e a t i n g would d e c o u p l e t h e m i c r o -wave c a v i t y as t h e r e was t o o much co p p e r i n t h e c a v i t y . In a l l c a s e s t h e sample s i z e was q u i t e s m a l l i n o r d e r t o f i t i n t o the s m a l l s t a c k s o f t h e T E 1 0 2 c a v i t y , and t h e EPR s i g n a l on a s a t u r a t e d system was not l a r g e enough. The more s u c c e s s f u l h i g h power system w i l l be d e s c r i b e d n e x t . 3.1 High Power S p e c t r o m e t e r The s p e c t r o m e t e r was c o n s t r u c t e d around an a v a i l a b l e V a r i a n X-Band V4502.100 kHz s p e c t r o m e t e r c i r c u i t . The f i n a l v e r s i o n had o n l y t h e magnet power s u p p l y , the k l y s t r o n power s u p p l y and t h e A.F.C. c i r c u i t r y unchanged. The o v e r a l l b l o c k diagram i s shown i n F i g 4 and t h e v a r i o u s m o d i f i c a t i o n s a r e d e s c r i b e d below. 3.2 The C a v i t y For the ENDOR e x p e r i m e n t , t h e V a r i a n w i d e - s a m p l e - a c c e s s c a v i t y was s e l e c t e d . T h i s i s a c y l i n d r i c a l wire-wound X-Band c a v i t y o p e r a t i n g i n t h e T E 0 1 3 mode. The w i r e w i n d i n g s t o p s e x c e s s i v e r a d i o f r e q u e n c y i n t e r f e r e n c e ( r f i ) . The l a r g e s t a c k s and 013 mode e n a b l e v e r y l a r g e samples t o be r a d i a t e d s i m u l t a n e o u s l y w i t h microwave ( H ^ and r f ( H 2 ) f i e l d s . The u s u a l sample H u b e was 8 cm h i g h and 10 mm o r more i n d i a m e t e r . I f no c o o l i n g i s d e s i r e d , the samples can go up t o 2 cm i n d i a m e t e r . Many m o d i f i c a t i o n s were made t o t h i s c a v i t y d u r i n g t h e s p e c t r o -E S R Experiment* Klystron AFC 44 KhZ [-copper box Microwave Bnc r ENDOR Experiment < Magneti Reid Control Temper-ature Control Modulptibn ! 1 j Lcopper box Detector copper shield To signal Generator cavity ± F M Modulation Signal Generator 200Watt Rf Amplifiers Servo System F M Modulation ENDOR Lock IN *1 (High Frequency) 10 K H Z Lock IN *2 (Low Frequency) 100 HZ AM to Held coils copper shield requency Counter 5^ D/A Converter X ilibration [Unit ^_Recader_ _ Block Diagram of Liquid ENDOR Spectrometer Figure 4. General Block Diagram of X-Band ENDOR and EPR Spectrometer: arrangements of detectors and frequency generators. 00 49 meter c o n s t r u c t i o n t o c o r r e c t poor d e s i g n and f a c i l i t a t e o p e r a t i o n . The o r i g i n a l d e s i g n was f o r ENDOR work a t 1 kW w i t h a 10% d u t y c y c l e , o r about 100 Watts o f heat d i s s i p a t i o n . F o r h i g h e r powers than t h i s , h o l l o w c o o l i n g caps were d e s i g n e d which f i t t e d v e r y s n u g l y o v e r t h e alum i n i u m c a v i t y ends. Tap water was c o n d u c t e d t h r o u g h t h e s e a t about 20 gph. To p r e v e n t c o r r o s i o n i n t h e p r e s e n c e o f mixed m e t a l s t h e s e caps were f l a s h e d l i g h t l y w i t h g o l d . The i n n e r c l e a r a n c e o f t h e s e caps from t h e r f c o i l s i s q u i t e c r i t i c a l and t e f l o n s t r i p s a r e used t o p r e v e n t s h o r t i n g o f t h e c o i l s . In t h e v e r y c o r r o s i v e a r e a n e a r t h e c o i l s ( h e a t , m o i s t u r e , f r o s t , mixed metal j u n c t i o n s ) even t h i s t e f l o n i n s u l a t i o n has o c c a s i o n a l l y f a i l e d . F o r c o r r o s i o n r e a s o n s t h e o r i g i n a l a l u m i n i u m c o i l r i n g s were a l s o r e p l a c e d w i t h g o l d - p l a t e d b r a s s . C o i l r i n g s were d e s i g n e d t o g i v e a c o i l impedance o f .34yH (a s i n g l e 2 - t u r n c o i l ) i n s t e a d o f t h e s u p p l i e d .lyH c o n f i g u r a t i o n (2 s i n g l e - t u r n c o i l s ) . The c a v i t y i s s u p p l i e d w i t h an a l u m i n i u m c o o l i n g s t a c k f o r s u p p o r t i n g t h e samples: a t low te m p e r a t u r e o p e r a t i o n t h e e x c e s s gaseous N 2 r e f r i g e r a n t c o o l s t h i s s t a c k and c o o l s the c a v i t y end, d e s t r o y i n g c o m p l e t e l y t h e v e r y h i g h Q ( W O , 0 0 0 ) and e x p l o s i v e l y f r e e z i n g t h e c o o l i n g w a t e r . T h i s s t a c k was r e b u i l t o ut o f H y s o l , an i n s u l a t i n g p l a s t i c . V a r i a n : s u p p l i e d t h e c a v i t y w i t h t h e i n n e r end p l a t e s s e c u r e d r a t h e r p o o r l y w i t h Duco cement. When t h i s g l u e e v e n t u a l l y f a i l e d , the p l a t e s were r e - f a s t e n e d w i t h s m a l l b r a s s b o l t s w i t h no d i f f e r e n c e i n r f i . The r f c o i l s c o n n e c t o r s were r e i n s t a l l e d on th e f r o n t t o m i n i m i z e t h e d i s t a n c e t o t h e t u n i n g c a p a c i t o r : t h e modula-t i o n c o i l s were then r e v e r s e d (and r e b u i l t ) t o p l a c e t h e i r c o n n e c t i o n s as f a r as p o s s i b l e f r o m t h e c a p a c i t o r . Copper r f s h i e l d s were i n s t a l l e d on both t h e f r o n t and back o f 50 t h e c a v i t y ; t h e s e a r e v i s i b l e i n F i g 5. These a l s o m i n i m i z e t he microwave l e a k a g e t h r o u g h t h e wire-wound c e n t r a l p r o t i o n , and wedge t h e c a v i t y i n p l a c e t o p r e v e n t motion and a c t as grounds t o t h e magnet p o l e p i e c e s f o r s p u r i o u s r f . The c a v i t y i s r f e l e c t r i c a l l y i n s u l a t e d f r o m t h e wave-guide by a s t a i . n l e s s - s t e e l - i n - h y s o l wave-guide s e c t i o n , u s i n g p l a s t i c g a s k e t s . I t i s s e c u r e d t o the wave g u i d e w i t h t e f l o n screws. S i m i l a r i n s e r t s a r e used i n v a r i o u s p l a c e s i n the wave g u i d e s t o s t o p r f f l o w . The r f c o i l s a r e a d e q u a t e l y c o o l e d by c o n d u c t i o n f r o m t h e water c o o l e d c a p a c i t o r l e a d t u b e s and by a i r c o n v e c t i o n , a l t h o u g h t h e t e f l o n i n s u l a t i n g s l e e v e s have m e l t e d once i n 3,000 hours o f o p e r a t i o n . The s t a c k s , end p l a t e s , c o i l s , and i n s e r t s must be a l i g n e d c a r e f u l l y d u r i n g r e a s s e m b l y w i t h a t e f l o n i n s e r t made f o r t h i s purpose. The T E 0 1 3 mode g i v e s 3 a r e a s o f h i g h microwave f i e l d ; o n l y 2 o f t h e s e a r e a s a r e i n an a r e a o f both h i g h f i e l d m o d u l a t i o n and h i g h r f f i e l d . The r f c o i l s must be c o n n e c t e d and a l i g n e d so t h a t H Q, 1^ and H 2 a r e m u t u a l l y p e r p e n d i c u l a r t o w i t h i n a few d e g r e e s : a t t h e sample s m a l l non-homogeneity o f H 2 i s n o t n o t i c e a b l e . The r f c o i l s were used as s u p p l i e d f o r some t i m e ; but w i t h a v e r y s i m p l e m o d i f i c a t i o n d o u b l e t h e r f f i e l d was o b t a i n e d a t t h e sample: new c o i l s were c o n s t r u c t e d o u t o f 1.6 mm n i c k e l - p l a t e d c o p p e r bent as shown i n F i g 5 t o l e a d t he r f w i r e s v e r y c l o s e t o t h e dewar. T h i s i s c a l l e d t h e "squeezed c o i l " c o n f i g u r a t i o n ( F i g 5 ) . The bent w i r e s - a r e i n s u l a f r o m t h e end p l a t e s by t e f l o n g a s k e t s w i t h g r o o v e s , f r o m t h e i r e n t r a n c e c h a n n e l s w i t h t e f l o n s l e e v e s , and from t h e end caps by c a r e f u l trimming a f t e r assembly. T h e r e i s some d i f f i c u l t y i n s e r t i n g t h e dewar, but once F igure 5. Squeezed r f c o i l s of c a v i t y . The Te f lon i n s u l a t o r s and copper r f s h i e l d s are a l so v i s i b l e . 52 p r o p e r l y i n s t a l l e d , t h e s e c o i l s w i l l not a l t e r t h e c a v i t y q u a l i t y v e r y much and e f f e c t i v e l y d o u b l e t h e a v a i l a b l e r f power. T h i s has been the l e a s t e x p e n s i v e m o d i f i c a t i o n f o r o b t a i n i n g a l a r g e r H 2 f i e l d a t t h e sample. I t f o l l o w s . . . t h a t t h e r f i from t h e c i r c u i t r y e x t e r n a l t o t h e c a v i t y i s l e s s w i t h t h e same i n t e r n a l H 2 f i e l d . The c a v i t y i s grounded i n s e v e r a l p l a c e s t o the magnet p o l e p i e c e by c o p p e r s t r a p s . D u r i n g o p e r a t i o n i t i s l i g h t l y p r e s s u r i z e d w i t h d r y N 2 t o e l i m i n a t e i n t e r n a l w a ter c o n d e n s a t i o n . A f t e r s t a b i l i z a t i o n o f t h e w a t er and r f - h e a t i n g t e m p e r a t u r e s , f r e q u e n c y d r i f t i s m i n i m a l , and t h e r e has been no need f o r a f i e l d - f r e q u e n c y l o c k . The epoxy w a l l s a r e e a s i l y p e n e t r a t e d by f i e l d m o d u l a t i o n , so t h a t t h e one low f r e q u e n c y m o d u l a t i o n c o i l can be used f o r both the 100 kHz normal EBR o p e r a t i o n and t h e wide range o f low f r e q u e n c y f i e l d - m o d u l a t i o n s (18 t o 1000 Hz) used f o r the ENDOR e x p e r i m e n t . 3.3 I n t e r n a l F i e l d Measurements The s t r e n g t h o f H Q does not have t o be measured i n s i d e t h e c a v i t y : t h e m a g n e t i c f i e l d i s m e r e l y swept u n t i l . E P R . i s . o b t a i n e d . • With a w i r e wound c a v i t y t h e p e n e t r a t i o n o f H Q i s e x c e l l e n t , and t h e d i f f e r e n c e o u t s i d e and i n s i d e t h e c a v i t y i s l e s s than 1 Gauss. The s t r e n g t h o f the microwave f i e l d , Hl must be f o u n d f o r or OC a p p l i c a t i o n o f e q u a t i o n g l } To measure H : t h e method o f C o p e l a n d ' was employed. In a c a v i t y o f h e i g h t h, volume V, c r o s s s e c t i o n a l a r e a , c o n t a i n i n g p h a l f ^ w a v e l e n g t h s (6 f o r a TE013 mode) a t a r e s o n a n t f r e q u e n c y f t h e microwave f i e l d i s g i v e n by: 53 8Q LP.K*10" 2-H l' = f V [ l + ( h / P A ) 2 ] C 5 0 ] where Q L i s t h e q u a l i t y f a c t o r w i t h a l o a d , K i s an unknown c o r r e c t i o n f a c t o r f o r f i e l d c o n c e n t r a t i o n by t h e dewar and P. i s t h e i n c i d e n t k l y s t r o n power. With t h e d i m e n s i o n s o f t h i s l a r g e c a v i t y H 2 = 3 . 8 x l O " 8 Q L P . . The q u a l i t y f a c t o r was- measured w i t h a de Mornay wavemeter t o o b t a i n t h e "O] Aco; f r e q u e n c y w i d t h a t h a l f - p o w e r (Q = ^°-) as shown i n T a b l e 2. TABLE 2 - CAVITY QUALITY MEASUREMENTS  C a v i t y C o n d i t i o n 0^  Empty > 20,000 Squeezed P o s t s 7,000 w Dewar 4,700 Sample ( i n o i l ) 1,395 L i q u i d C r y s t a l 1,260 T h i s l e a d s t o Hi = .0073 /PT [51] f o r t h e l o a d e d c a v i t y (power i n mW). The most i m p o r t a n t f i e l d , H 2 f r o m t h i s and e q u a t i o n p i ] must th e n be around 20 Gauss o r g r e a t e r . The magnitude o f H§'in t he c a v i t y was measured i n two ways;' d a y - t o -day measurement was pe r f o r m e d w i t h a 5 - t u r n o r i e n t a t e d c o p p e r c o i l c onnec-t e d i n a s h i e l d e d brass-'tube t o a low-wattage l i g h t b u l b which was 54 s e l e c t e d t o j u s t g i v e a glow a t the s t a n d a r d o p e r a t i n g r f f i e l d 12 G a u s s ) . S i n c e t h e t u b e was t h e same s i z e as a sample t u b e , t h i s c o u l d be used t o check on t h e o r i e n t a t i o n o f t h e H 2 f i e l d , t h e t u n i n g o f t h e tank c i r c u i t and t h e e l e c t r i c a l c o n n e c t i o n s o f t h e r f systems. A c c u r a t e H 2 measurements were o b t a i n e d w i t h a c a r e f u l l y c on-s t r u c t e d and c a l i b r a t e d 3 t u r n c o i l , and l e a d w i r e , tuned t o around 55 MHz. PP Peak t o peak v o l t a g e , V was measured on a h i g h f r e q u e n c y o s c i l l o s c o p e . For t h i s measurement and t h i s c o i l II _ ,,PP,„ _ ( v P P -x 10> \ _ V P P x (4.69) r, ? 1  H 2 ~ H / 4 " p A 2 7 f | / 4 f(MHz) ' [ 5 2 ] where N = 3 t u r n s and A i s t h e c o i l a r e a . T h i s c o i l was c a l i b r a t e d a g a i n s t a n o t h e r known c o i l and used t o o b t a i n t h e measurements o f T a b l e 3. TABLE 3. H? measurements i n t h e r o t a t i n g frame (G) A m p l i f i e r i n p u t NMR f r e q u e n c y (mV) 10 MHz 15 MHz 20 MHz 180 10,6 8.9 7.3 250 14.7 12.4 10.2 500 26.3 22.2 18.3 1000 33 28 23 Beyond 400 mV, t h e p i c k u p i n t h e r e s t o f t h e system i s u n c o n t r o l l a b l e , so t h a t an e f f e c t i v e maximum f o r H 2 i s around 15 Gauss u n t i l b e t t e r s h i e l d i n g can be d e s i g n e d . 55 3.4 Radio Frequency System The r f system c o n s i s t s o f a Marconi 2002AS s i g n a l g e n e r a t o r , 2 E l e c t r o n i c N a v i g a t i o n I n d u s t r i e s s o l i d s t a t e 3100L 100 W a m p l i f i e r s w i t h c o u p l e r s , and an r f t r a n s f o r m e r c o n n e c t e d t o t h e tank c i r c u i t . A l l o f t h e s e components a r e w e l l d e s i g n e d f o r ENDOR a p p l i c a t i o n , and have p e r f o r m e d w i t h o u t breakdown f o r o v e r 3,000 h o u r s . The Marconi i s fm modulated by t h e P.A.R. 121 P.S.D., a m p l i f i e d by a H e w l e t t P a c k a r d 450A 60W a u d i o a m p l i f i e r t o g i v e 50 kHz d e v i a t i o n on t h e r f s i g n a l . The Marconi i s swept by a v a r i a b l e speed motor and gearbox g i v i n g a sweep r a t e , o f from 0.01 t o 6 MHz/min. The E.N.I, a m p l i f i e r s r u n r a t h e r h o t i n t h e i r s h i e l d e d c o p p e r c a g e , b u t o t h e r w i s e have p e r f o r m e d v e r y w e l l . They have ( a c c i d e n t a l l y ) been s h o r t e d a t t h e o u t p u t s e v e r a l t i m e s w i t h no e f f e c t , e x a c t l y as s p e c i f i e d by t h e m a n u f a c t u r e r s . They do run ' r f hot' w i t h t h e p r e s e n t c a v i t y l o a d , which makes them dangerous d u r i n g o p e r a t i o n , but run f a r l o n g e r and c o o l e r than t h e comparable tube t y p e I . F . I , a m p l i f i e r s t h a t 38 have been used e l s e w h e r e . The r f t r a n s f o r m e r s a r e s i m i l a r t o t h o s e d e s c r i b e d by 37 S c h m a l b e i n and a r e shown i n F i g 6 w i t h t h e i r s c h e m a t i c . They a r e c o n s t r u c t e d o f heavy gauge-wire wound s y m e t r i c a l l y on l a r g e f e r r i t e r i n g s : a l l e l e c t r i c a l c o n n e c t i o n s a r e hard s o l d e r e d o r b o l t e d . In such a d e v i c e , t h e l e n g t h o f w i r e , L, must be such t h a t ^ » L where X i s the wa v e l e n g t h a t t h e o p e r a t i n g f r e q u e n c y . In ENDOR th e u s u a l w a v e l e n g t h i s X - 60 m, so t h i s r e q u i r e m e n t i s s a t i s f i e d i n th e 5 t u r n c o i l used. The number o f t u r n s does n o t change t h e c h a r a c t e r -56 16: i Figure 6. (a) Four-to-one r f transformer and (b) Schematic diagram of 16:1 r f transformer. 57 i s t i c m a t c h i n g r a t i o , so t h e r e i s a c h o i c e o f 4:1, 16:1, and 64:1 o n l y . Optimum matching was o b t a i n e d w i t h a 16:1 r a t i o . - - - T h e a c t u a l impedance.of the tank c i r c u i t a t r a d i o f r e q u e n c i e s i s d i f f i c u l t t o measure e x a c t l y . I t was e s t i m a t e d t o be about 700 fi, so t h a t a 12:1 r a t i o would have been b e s t w i t h t h e 50 o u t p u t o f t h e a m p l i f i e r s . These t r a n s f o r m e r s run h o t enough t o have c r a c k e d once, and an o i l - f i l l e d immersion b a t h has been d e s i g n e d t o c o n t a i n them. The n o n - l i n e a r s i g n a l - g e n e r a t o r - b a n d i s swept by t h e v a r i a b l e speed motor: to g i v e a l i n e a r sweep, t h e r f s i g n a l i s d i v i d e d b e f o r e g o i n g t o t h e a m p l i f i e r s . a n d ' p a r t i s s e n t t h r o u g h a f r e q u e n c y c o u n t e r ( H e w l e t t P a c k a r d 5246L) D i g i t a l - A n a l o g c o n v e r t e r (HP-580A) t o - t h e . X a x i s o f the XY r e c o r d e r (HP 7005-B). When t h e 1 o r 0.1 MHz d i g i t f l i p s t o z e r o a s p e c i a l l y c o n s t r u c t e d c a l i b r a t o r sends a p u l s e t o the Y a x i s o f the"XY'.' T h i s can be o v e r r i d d e n by hand t o c a l i b r a t e a t s p e c i a l p o i n t s . T h i s arrangement g i v e s an a u t o m a t i c a l l y c a l i b r a t e d l i n e a r X sweep o f the f r e q u e n c y . T h e r e i s not enough s e n s i t i v i t y i n t h i s f r e q u e n c y c o u n t e r t o r e a d the i n - c a v i t y r f f r e q u e n c y v i a a p i c k u p c o i l as has been done by o t h e r 38 w o r k e r s . T h i s i s no r e a l l o s s , as t h e f r e q u e n c y c o u n t e r removes o n l y about 5% o f t h e i n p u t s i g n a l - v o l t a g e . With t h i s p r o c e d u r e , one can e a s i l y c a l i b r a t e s p e c t r a t o ± 20 kHz and w i t h some d i f f i c u l t y t o + 5 kHz. 3.5 Servo System and C a p a c i t o r To match t h e a m p l i f i e r s more p r e c i s e l y t o t h e c a v i t y r f c o i l , i t i s n e c e s s a r y t o use a h i g h v o l t a g e , l a r g e - r a n g e c a p a c i t o r i n a tank c i r c u i t w i t h t h e c o i l . To tune t h i s (low' Q) c i r c u i t c o n t i n u o u s l y , some 58 s o r t o f r a p i d - r e s p o n s e s e r v o - s y s t e m must be d e s i g n e d . Manual t u n i n g i s p o s s i b l e , but n o t f o r l o n g s c a n s . The l a r g e r f f i e l d i s g e n e r a t e d i n a tank c i r c u i t , c o n s i s t i n g o f t h e 2 - t u r n c a v i t y c o i l and a ITT J e n n i n g s v a r i a b l e vacuum c a p a c i t o r (19 t o 2500 p f ) . A t u n a b l e f r e q u e n c y range o f 5 MHz t o 26 MHz i s a v a i l a b l e : beyond t h e r a n g e , t h e H 2 f i e l d d r o p s o f f so r a p i d l y t h a t no ENDOR i s v i s i b l e w i t h o n l y 200 Watts o f r f power. The c a p a c i t o r i s c o o l e d by c o n d u c t i o n from t h e w a t e r - c o o l e d 1 cm co p p e r p i p e which e l e c t r i c a l l y connects t h e c a p a c i t o r t o the r f c o i l . These two p i p e s a r e " s h o r t e d " , t o each o t h e r by a moving water s t r e a m , but t a p water does not co n d u c t r f . A p p r o x i m a t e l y 40 t o 80 amperes f l o w i n t h i s tank c i r c u i t , but t h e water and a i r c o n d u c t i o n s e r v e t o keep t h e c a p a c i t o r c o o l . The c a p a c i t o r and i t s water p i p e s a r e s e a l e d i n a c o p p e r box t o m i n i m i z e r f l e a k a g e . A s i g n a l i s taken d i r e c t l y o f f t h i s tank c i r c u i t t h r o u g h two r e d u c t i o n r e s i s t o r s ( 1 0 5 a) then t h r o u g h a f i r s t s t a g e fm d e t e c t o r and out o f t h e c o p p e r box. The d e t e c t e d s i g n a l i s l e d t o a PAR 122 L o c k - I n a m p l i f i e r . o p e r a t i n g a t t h e r f m o d u l a t i o n f r e q u e n c y . A f t e r a m p l i f i c a t i o n and phase s h i f t i n g , t h i s s i g n a l d r i v e s a s e r v o a m p l i f i e r (AST-10W s o l i d s t a t e o r a s i m i l a r system c o n s t r u c t e d h e r e , d e p e n d i n g on t h e s e r v o motor i n u s e ) ; The a m p l i f i e r — d r i v e n motor i n t u r n d r i v e s t h e caged c a p a c i t o r t h r o u g h a 60:1 gearbox (Link. i A v i a t i o n ) a c l u t c h , a d i f f e r e n t i a l u n i t ( t o p r o t e c t t h e c a p a c i t o r ) and a r f i s o l a t i o n r o d . Most o f t h e s e u n i t s a r e v i s i b l e i n F i g 7, showing t h e e x p l o d e d - v i e w c a v i t y and t h e s e r v o system. A t low f r e q u e n c i e s a l o t o f s e r v o g a i n i s n e c e s s a r y ; near t h e t o p o f an ENDOR scan t h e whole e l e c t r o - m e c h a n i c a l system overcompensated. 59 Figure 7. Servo System and cavity modif icat ions: The capacitor and i t s servo drive system (shielded) are on lower l e f t . Exploded view of the cav i ty , cooling system and sample dewar on r igh t . A 16:1 r f transformer is also shown. 60 To c o r r e c t t h i s , t he s e r v o system g a i n i s a d j u s t e d s l i g h t l y w i t h a v a r i a b l e r e s i s t o r i n t h e i n i t i a l d e t e c t i o n c i r c u i t d u r i n g a s c a n . E x p e r i m e n t s showed t h a t a d i r e c t s i g n a l p i c k u p i n t h e tank c i r c u i t i t s e l f gave f a r more s e r v o s t a b i l i t y on t h i s system t h a n the 38 method o f a p i c k u p c o i l n e a r t h e c a v i t y as used by A l l e n d o e r f e r . The p i c k u p c o i l i s s t i l l used f o r a v i s u a l i n d i c a t i o n o f t h e r f f i e l d s t r e n g t h . The m e c h a n i c a l system can be m a n u a l l y d r i v e n t h r o u g h d i f f e r e n -t i a l g e a r s f o r e a s i e r s e r v o t u n i n g . In t h i s system t h e r e i s enough s e r v o g a i n a v a i l a b l e t o tune anywhere on t h e d e t e c t e d tank c i r c u i t r e s o n a n c e and t h e s e r v o i s u s u a l l y o s c i l l o s c o p e - t u n e d t o a minimum r f p i c k u D a t the m i c r o -wave.diode. I t was found t h a t t h i s s e r v o s e t t i n g v a r i e d from day t o day, and a l s o v a r i e d w i t h t h e r f m o d u l a t i o n f r e q u e n c y . S i n c e t h e motor and g e a r -box o f t e n a r e r f h o t , a s e c o n d a r y copper cage was c o n s t r u c t e d t o house t h e s e components. 3.6 Microwave and D e t e c t i o n System The microwave b r i d g e was c o n s t r u c t e d f r o m s t a n d a r d X--band de Mornay wave g u i d e components. These a r e grounded t o t h e magnet," t o each o t h e r , and t h e c o p p e r boxes, and p e r i o d i c a l l y d i v i d e d w i t h i n s u l a t i n g segments as d e s c r i b e d above, t o minimze t h e e f f e c t o f r e - e m i t t e d r f . E x p e r i m e n t s showed t h a t t h e r e was a r e m a r k a b l e improvement i n S/N by p l a c i n g t h e d e t e c t o r d i o d e and e v e n t u a l l y t h e k l y s t r o n i n c o p p e r boxes, grounded t o t h e same c h a s s i s as t h e l o c k - i n a m p l i f i e r system. These boxes were o i l and w a t e r - t i g h t ( f o r k l y s t r o n c o o l i n g ) and as r f i f r e e as p o s s i b l e . A f t e r much e x p e r i m e n t a t i o n , the d e t e c t i o n system s e l e c t e d 61 was a S h o t t k y - b a r r i e r microwave d i o d e . A t t h e f r e q u e n c i e s used f o r t h e m o d u l a t i o n s , t h e s e have a s i g n a l - t o - n o i s e r a t i o a l m o s t t r i p l e t h a t o f a normal d i o d e . They have a v e r y low o p e r a t i n g l i f e t i m e and a wide v a r i a t i o n i n i n d i v i d u a l d i o d e q u a l i t y so t h a t a c a r e f u l s e l e c t i o n o f t h e d i o d e i n use must be made. The s i g n a l i s a m p l i f i e d and s e n t v i a a s h o r t l e a d t o t h e f i r s t l o c k - i n a m p l i f i e r . The p r e a m p l i f i e r i s a l s o i n t h e c o p p e r c a g e , and has s e v e r a l r f s h u n t i n g c a p a c i t o r s i n i t t o e l i m i n a t e s p u r i o u s r f i . They do a v e r y poor j o b : any r f i t h a t has p e n e t r a t e d t h e c o p p e r cage t h i s f a r i s n o r m a l l y v i s i b l e on t h e s p e c t r a . U n f o r t u n a t e l y because, o f t h e r f modula-t i o n d i o d e p i c k u p o f r f i resembles' an ENDOR s i g n a l i n a11 r e s p e c t s . 3.7 Magnet and S h i e l d i n g A t p r e s e n t a c u r r e n t - c o n t r o l l e d V a r i a n 12" magnet i s i n use. 38 T h i s e l i m i n a t e s t h e g r e a t problem o f r f i i n a H a l l p r o b e , b ut r e q u i r e s l o n g e r warm up t i m e s t o e l i m i n a t e f i e l d d r i f t . The ma g n e t i c f i e l d i s scanned by a u n i t c o n s t r u c t e d i n t h i s department,.and l o n g term d r i f t i s e l i m i n a t e d by u s i n g narrow scan w i d t h s (3 Gauss o r l e s s ) . So f a r t h e r e has been no need f o r a f i e l d - f r e q u e n c y l o c k on t h e system i f s u f f i c i e n t t i m e i s a l l o w e d f o r magnet s t a b i l i t y . T h e r e i s some r f i i n the power s u p p l y o f t h i s magnet, but i t i s a t an a c c e p t a b l e l e v e l w i t h t h e 200 W a m p l i f i e r s . The tremendous advantage o f t h i s magnet i s t h a t i t i s sh e a t h e d i n p a i n t e d c o p p e r . T h i s was s c r a p e d c l e a n and a c y l i n d r i c a l c o p p e r cage clamped f i r m l y a l l t h e way around t h e magnet, e n c l o s i n g t h e c a v i t y and c a p a c i t o r , and a l m o s t e l i m i n a t i n g o u t s i d e r f i . F u r t h e r m o r e , b o t h t h e 50 W 62 and 100 W ENI a m p l i f i e r s , which run " r f h o t " , f i t under t h e magnet yoke, and can be e n c l o s e d i n a c o p p e r cage as w e l l . A l l o f t h e c o p p e r cages can t h u s be grounded t o t h e magnet, t h e l a r g e s t mass o f metal i n t h e system. T h i s has proved t o be t h e b e s t r f ground a v a i l a b l e , but t t i s by no means p e r f e c t . T h e r e a r e two t y p e s o f r f i i n a h i g h r f power system: t h a t conduc-t e d a l o n g m e t a l l i c pathways, and t h a t which i s b r o a d c a s t t h r o u g h space t o a n d f r o m components a c t i n g as a n t e n n a e . F o r b r o a d c a s t p u r p o s e s , a h o l e i n t h e s h i e l d up t o 10 cm; i n d i a m e t e r has p r o v e d t o be r f l e a k - t i g h t a t t h e f r e q u e n c i e s i n use, so that-many o f t h e systems t h a t must l e a d i n t o t h e mag-n e t - c a g e e n v i r o n m e n t can j u s t pass t h r o u g h h o l e s i n t h e cage. These i n c l u d e f a n s , magnet-and r f - c o o l i n g w a t e r , waveguide, s a m p l e - c o o l i n g dewar, t e m p e r a t u r e c o n t r o i l l e a d s , c o u p l i n g r o d s and magnet power. Many o f t h e e l e c t r i c a l systems have l a r g e amounts o f c o n d u c t e d r f i n them and t h e s e a r e c a r e f u l l y f i l t e r e d a t t h e i n s i d e edge o f the c a g e . These i n c l u d e r f a m p l i f i e r s ( f i l t e r e d from t h e l i n e v o l t a g e ) , m o d u l a t i o n c o i l s and s e r v o m e c h a n i c a l d r i v e t o t h e c a p a c i t o r . T h i s cage has proved t o be t h e most e f f e c t i v e weapon a g a i n s t r f i , and has c r e a t e d - d a y - t o - d a y r e p r o d u c i b i l i t y w i t h no 'cable-wavincj' o r e n v i r o n m e n t a l c a p a c i t a n c e e f f e c t s , as opposed t o a 38 s p e c t r o m e t e r w i t h s h i e l d e d c a b l e s where t h e s e e f f e c t s can be e l i m i n a t e d b u t a r e l a r g e a t t i m e s . 3.8 Temperature C o n t r o l A V a r i a n E257 t e m p e r a t u r e c o n t r o l u n i t i s used, w i t h a l a r g e dewar. S i n c e t h e r f c o i l w i r e s a r e o u t s i d e t h e dewar, t h e r e i s n o t much thermal s t r e s s on t h e system; however r f i i s o f t e n p i c k e d up by t h e t h e r m i s t o r which i s a l m o s t i n t h e microwave c a v i t y . Rf c a p a c i t o r s were i n s t a l l e d i n t h e E257 a t c i r c u i t p o i n t s recommended by V a r i a n , and t w i s t e d 63 w i r e was used f o r t h e l e a d s , t o c u t down on r f i . A t p r e s e n t a l a r g e r t e m p e r a t u r e e r r o r (about 2°C) o c c u r s f r o m t h e use .of t h e l o w - c a p a c i t y h e a t e r s u p p l i e d * b y . V a r i a n w i t h t h i s l a r g e d e w a r i t h a n f r o m r f i ( l e s s t h a n 0 . 5 ° C ) . A c c u r a t e t e m p e r a t u r e s a r e measured w i t h a c o p p e r - c o n s t a n t a n thermo-c o u p l e . T h i s system i s v e r y poor a t low t e m p e r a t u r e s s i n c e v e r y l a r g e q u a n t i t i e s o f both gaseous N 2 and r e f r i g e r a n t - l i q u i d N. 2~are u s e d . From -100 to +100 °C i t i s q u i t e a c c e p t a b l e ( w i t h a v a r i a t i o n of, ± 1 ° C ) , but o u t s i d e t h i s r a n g e , a r e - c y c l i n g system s h o u l d be used. The t r a n s f e r dewar and r e f r i g e r a n t dewar a r e n o r m a l l y r f "hot" d u r i n g a scan and must remain immobile t o m i n i m i z e p i c k u p . 3.9 EPR D e t e c t i o n The EPR s p e c t r a were always d e t e c t a b l e on t h e system as modi-f i e d , but t h e com p a r i s o n EPR s p e c t r a were u s u a l l y o p t i m i z e d on a V a r i a n E-3100 kHz s p e c t r o m e t e r a v a i l a b l e i n t h i s department. Optimum 100 kHz d e t e c t i o n on t h e ENDOR s p e c t r o m e t e r i s d i f f i c u l t as few o f t h e m o d u l a t i o n d e p t h s o r d i a l s e t t i n g s a r e ^ c a . l i b r a t e d . i n Gauss. The c o m p l e t e ENDOR s p e c t r o m e t e r w i t h placement o f s h i e l d i n g boxes and t h e i n t e r c o n n e c t i o n s i s shown i n F i g 8. The o p e r a t i o n o f t h e EPR e x p e r i m e n t ( t o p o f F i g u r e 8) w i l l be d e s c r i b e d n e x t . The k l y s t r o n ( V a r i a n 350 mW 153C) i s modulated by a V a r i a n 4500-10A microwave c o n t r o l u n i t , w i t h t h e A.F.C. f r e q u e n c y s h i f t e d t o 43 kHz t o a v o i d o t h e r f r e q u e n c i e s i n t h e ENDOR e x p e r i m e n t . ESR a t 100 kHz i s d e t e c t e d w i t h e i t h e r t h e V a r i a n 4560 d e v i c e o r a s p e c i a l l y c o n s t r u c t e d 100 kHz power s u p p l y u n i t combined w i t h a H e w l e t t Packard 121 L o c k - i n 64 a m p l i f i e r . The s i g n a l i s e i t h e r p r e s e n t e d on a Mosl e y 680 S t r i p c h a r t r e c o r d e r o r t h e HP - XY r e c o r d e r . Low-frequency EPR i s o b t a i n e d w i t h t h e HP 121 o p e r a t i n g a t 18-400 kHz. M o d u l a t i o n from t h e L o c k - i n i s a m p l i f i e d by a H e w l e t t Packard 450A 60W a u d i o a m p l i f i e r and l e d t o t h e f i e l d c o i l s t h r o u g h a s e r i e s o f h i g h - f r e q u e n c y choke c o i l s l o c a t e d on t h e co p p e r cage w a l l s . The EPR s i g n a l i s l e d from t h e microwave d i o d e t o t h e L o c k - i n and t h e n c e t o t h e r e c o r d i n g system v i a v e r y s h o r t l e a d s . Microwave power up t o 300 mW f o r t h e ESR e x p e r i m e n t i s measured -witha H e w l e t t P a c k a r d 431-C Power meter. With t he h i g h s e n s i t i v i t y o f t h e d e t e c t i o n system a l l EPR s p e c t r a c o u l d be o s c i l l o s c o p e - d i s p l a y e d f o r e a s y o p t i m i z a t i o n . A t a l l m o d u l a t i o n f r e q u e n c i e s , t h e s i g n a l - t o - n o i s e r a t i o f o r t h e Varian- s t a n d a r d w h i t e p i t c h sample"was 'about 20 to 1. A t low f r e q u e n c i e s t h e p e n e t r a t i o n o f t h e c a v i t y by t h e f i e l d m o d u l a t i o n was b e s t ; a t h i g h e r f r e q u e n c i e s t h e a m p l i f i e r n o i s e was much lower a l t h o u g h p e n e t r a t i o n was poor; t h e s e two e f f e c t s c a n c e l each o t h e r so t h a t good d e t e c t i o n i s p o s s i b l e a t a l l f r e q u e n c i e s . 3.10 ENDOR O p e r a t i o n D u r i n g an ENDOR s c a n , t h e p r e - a m p l i f i e d s i g n a l i s l e d f i r s t v i a a s h o r t l e a d t o a PAR 122 a m p l i f i e r d e t e c t i n g a t a h i g h f r e q u e n c y and th e n t o t h e low f r e q u e n c y PAR 121 L o c k - i n used i n EPR and f i n a l l y t o t h e XY r e c o r d e r , o p e r a t i n g a t a h i g h a m p l i f i c a t i o n o f 10 mV/inch. The f i l t e r i n g o f t h e EPR L o c k - i n i s r a i s e d t o a 3 sec time c o n s t a n t from t h e EPR v a l u e ( . 1 s e c ) tc e l i m i n a t e much s p u r i o u s n o i s e . O t h e r c o n n e c t i o n s a r e as shown i n F i g 8. r AFC. PS. a> UJ QJ 8-UJ r Klystron Cooling Woter Cut Out —r Klystron P.S. A.FC. 8-10 GH€ Klystron Detcurr. Copper Box Meter Copper Box < or £ O Magnet Scon Unit To xy Recorder, x To Cavity \Afater Mocte Sweep To Scope EPR Control unit Marian V 4 5 C O ,"lb Scope Cavity Cooling Water Cutout MOV i power Signal Generator Marconi 2002AS. Scope Selection |To Coils a t O O k H f U.B.C. Liquid ENDOR Spectrometer Figure 8. Detailed Block Diagram of high-power liquid ENDOR Spectrometer, showing all component connections. 66 The s e a r c h f o r ENDOR enhancement i n v o l v e s i t e r a t i o n o f s e v e r a l b a s i c s t e p s . S e v e r a l o f t h e s e can be e l i m i n a t e d b e f o r e s t u d y o f an unknown sample i f t h e s p e c t r o m e t e r i s f i r s t o p t i m i z e d on a sample known t o g i v e ENDOR. I n i t i a l l y , a l l t h e s p e c t r o m e t e r components a r e warmed up f o r a t l e a s t t h r e e hours t o a c h i e v e good t e m p e r a t u r e s t a b i l i z a t i o n . Frequency s h i f t s i n t h e c a v i t y caused by d r i f t can be s e v e r e enough t o s h i f t t h e s p e c t r o m e t e r c o m p l e t e l y o f f t h e e l e c t r o n r e s o n a n c e p o i n t d u r i n g a s c a n ; such t e m p e r a t u r e d r i f t s w i l l o c c u r upon t u r n i n g on t h e r f f i e l d , u n l e s s t h e r f h e a t i n g e f f e c t and t h e c a v i t y water c o o l i n g a r e a l l o w e d t o e q u i -l i b e r a t e f o r t h i s l e n g t h o f t i m e . A sample i s o u t g a s s e d i n a 30 cm by 10 mm q u a r t z sample t u b e ; the c o n c e n t r a t i o n i s a d j u s t e d t o g i v e optimum power s a t u r a t i o n a t 100 kHz (on t h e E-3) a t l e s s than 50 mW. I f n e c e s s a r y t h e t e m p e r a t u r e (and hence the v i s c o s i t y ) a r e changed u n t i l t h i s o c c u r s . Power s a t u r a t i o n has been f o u n d t o v a r y w i t h t h e f i e l d - m o d u l a t i o n f r e q u e n c y , so t h a t t h i s optimum sample i s r e c h e c k e d a t the low ENDOR m o d u l a t i o n f r e q u e n c y f o r s a t u r a t i o n . T h e r e a r e two t y p e s o f s a t u r a t i o n f o r s o l u t i o n s o f r a d i c a l s : both o f t h e s e have been o b s e r v e d h e r e . In t h e f i r s t t y p e a sample shows a c l e a r d e c r e a s e i n i n t e n s i t y w i t h i n c r e a s i n g microwave power beyond t h e s a t u r a t i o n maximum. In t h i s c a s e , optimum ENDOR i s o b t a i n e d from 1-2 dB above t h i s max-imum. In t h e second t y p e o f s a t u r a t i o n t h e i n t e n s i t y i n c r e a s e s c o n t i n u o u s l y w i t h power but t h e r a t e o f i n c r e a s e slows down a f t e r a c e r t a i n v a l u e o f t h e power. In t h i s , c a s e optimum ENDOR i s o b t a i n e d a t a power which would have g i v e n an i n t e n s i t y t w i c e t h a t which was o b s e r v e d a t t h e i n f l e c t i o n p o i n t , i f t h e l i n e a r p r o t i o n o f s a t u r a t i o n c u r v e i s e x t r a p o l a t e d beyond t h e i n f l e c t i o n p o i n t . In t h e t h i r d c a s e o f no s a t u r a t i o n b e h a v i o u r b e i n g o b s e r v e d , one ch o o s e s 67 a microwave power as l a r g e as p o s s i b l e t h a t w i l l not c a u s e e x c e s s i v e n o i s e a t t h e f i r s t s t a g e d e t e c t o r d i o d e . (ENDOR has been o b t a i n e d on t h i s i n s t r u m e n t a t 185 mw o f microwave power). D u r i n g t h e ENDOR exp e r i m e n t a l a r g e * h i g h l y - m o d u l a t e d microwave s i g n a l a r r i v e s a t t h i s d i o d e : i t i s q u i t e p o s s i b l e t o c l i p t h e d i o d e w i t h e x c e s s i v e power. A low f r e q u e n c y i s chosen f o r t h e f i e l d m o d u l a t i o n t o m i n i m i z e t h e n o i s e a t 60 c y c l e s i n t h e l o c k - i n a m p l i f i e r ; t h e f r e q u e n c y f o r m o d u l a t i o n o f t h e r f i s chosen t o be w e l l away from t h e A.F.C. f r e q u e n c y and t h e f i e l d m o d u l a t i o n f r e q u e n c y , and a l s o t o m i n i m i z e L o c k - i n n o i s e and maximize t h e ENDOR s i g n a l . T h i s i s u s u a l l y done on t h e known sample Wide v a r i a t i o n s i n ENDOR enhancement a r e o b t a i n e d w i t h s m a l l changes i n r f fm f r e q u e n c y ( f o r i n s t a n c e t h e r a t i o o f enhancement a t 11 kHz t o t h a t a t 13 kHz i s about 11:1). T h i s i s c a u s e d by t h e d i f f e r e n t band-pass a b i l i t i e s o f t h e L o c k - i n . a m p l i f i e r a t d i f f e r e n t f r e q u e n c i e s . The d e p t h o f f i e l d m o d u l a t i o n i s chosen t o broaden and c o n n e c t a l l o f t h e ESR l e v e l s (around 10 G a u s s ) , but not t o d e c r e a s e t h e signa.l i n t e n s i t y : i t i s p o s s i b l e t o use t o o much f i e l d m o d u l a t i o n and d e c r e a s e t h e ESR s i g n a l , e s p e c i a l l y on r a d i c a l s w i t h narrow;ESR s p e c t r a l w i d t h s such as TTBP. The depth o f t h e fm i s s e t a t t h e v e r y maximum (50 kHz) t o s e a r c h f o r unknown s i g n a l s and t h e n d e c r e a s e d o n l y f o r o b s e r v i n g v e r y s m a l l d i f f e r e n c e s i n c o u p l i n g s . The r f i s s e t a t t h e maximum power a v a i l a b l e t h a t has minimum r f i (about 250 w a t t s ) . The magnetic f i e l d i s then s e t a t t h e h i g h f i e l d extremum of t h e ESR s i g n a l , and i s n o r m a l l y on such a narrow scan t h a t any d r i f t o f t h e u n i t w i l l n o t s h i f t t h e s p e c t r o m e t e r o f f t h e s i g n a l . 68 For ENDOR t h e L o c k - i n a m p l i f i e r s a r e now c o n n e c t e d t o g e t h e r , and t h e phase o f the f i e l d m o d u l a t i o n a m p l i f i e r s h i f t e d ( u s u a l l y ^ 110° phase s h i f t a t low f r e q u e n c y ) . The s i g n a l g e n e r a t o r i s then s l o w l y swept (1 MHz/min) around t h e f r e e p r o t o n f r e q u e n c y u n t i l a s i g n a l i s o b t a i n e d , i f p o s s i b l e w i t h o s c i l l o s c o p e d i s p l a y . A t t h i s p o i n t t h e magnetic f i e l d , microwave power, t e m p e r a t u r e , depth o f f i e l d m o d u l a t i o n , phases o f both L o c k - i n d e t e c t o r u n i t s , and r f power a r e c y c l i c a l l y a d j u s t e d u n t i l a maximum s i g n a l enhancement i s o b t a i n e d . The l a r g e s t a d j u s t m e n t s a r e n o r m a l l y t o t h e f i e l d and power, but a l l o f t h e s e parameters v a r y from sample t o sample, and a s t a n d a r d g i v e s o n l y rough v a l u e s . The s p e c t r u m i s then scanned s l o w l y (.2 MHz/min) w i t h minimum depth o f fm and c a l i b r a t e d e v e r y 1 o r .1 M H z . a u t o m a t i c a l l y . 3.11 Sample P r e p a r a t i o n As shown by e q u a t i o n [ 2 5 ] , t h e optimum ENDOR enhancement s h o u l d o c c u r a t h i g h v i s c o s i t y and low t e m p e r a t u r e . These c o n d i t i o n s o f h i g h v i s c o s i t y can be met i n s e v e r a l s o l v e n t s such as n-heptane o r t o l u e n e j u s t above t h e i r f r e e z i n g p o i n t s . H o l d i n g a sample j u s t a t t h i s h i g h v i s c o s i t y p o i n t does n o t a l l o w much v a r i a t i o n o f o t h e r p a r a m e t e r s , and s o l v e n t s such as m i n e r a l o i l w i t h a h i g h v i s c o s i t y o v e r a wide range o f t e m p e r a t u r e s a r e p r e f e r a b l e . Some o f t h e e a r l i e r s t u d i e s were run i n n-heptane a t -91°C but t h e enhancement was n e v e r as g r e a t as t h a t i n m i n e r a l o i l . L a r g e samples a l s o i n c r e a s e t h e a b s o l u t e ENDOR s i g n a l s t r e n g t h as i t i s p r o p o r t i o n a l t o t h e EPR s i g n a l ( e q u a t i o n [ 2 1 ] ) . 69 A c c o r d i n g l y , samples were u s u a l l y p r e p a r e d i n l a r g e q u a r t z sample t u b e s (30 cm x 10 mm). The commonest s o l v e n t f o r ENDOR was h i g h l y o u t g a s s e d m i n e r a l o i l ( F i s h e r , r e a g e n t g r a d e ) . The g r e a t e s t d i f f i c u l t y was i n d i s s o l v i n g t h e s p e c i e s under s t u d y i n t h i s s o l v e n t : sometimes t h e r a d i c a l was p r e p a r e d i n t h e o i l , sometimes i t was taken i n w i t h a n o t h e r s o l v e n t . A m i n e r a l o i l sample can be o u t g a s s e d by s e v e r a l means. Pumping f o r 3 t o 4 days w i l l remove a l l d i s s o l v e d oxygen i f t h e r a d i c a l i s s t a b l e f o r t h i s l e n g t h o f t i m e . M i n e r a l o i l w i t h o u t r a d i c a l can be heated under vacuum t o o u t g a s i t , and used t h e r e a f t e r w i t h minimal expos-u r e t o a i r . U l t r a s o n i c v i b r a t i o n o f a p r e p a r e d sample works w e l l , as does e l e c t r o s t a t i c d e g a s s i n g ( u s u a l l y by s t r o k i n g t h e sample t u b e w i t h a d r y t i s s u e ) . Normal freeze-pump-thaw-pump t e c h n i q u e s work, b u t r e q u i r e from 12 t o 20 f r e e z i n g s t o o u t g a s c o m p l e t e l y . Fo r EPR t h e commonest s o l v e n t was n-heptane ( F i s h e r , r e a g e n t g r a d e ) d r i e d o v e r m o l e c u l a r s i e v e and o u t g a s s e d a f t e r t h e r a d i c a l was d i s s o l v e d . T h r e e t y p e s o f samples were s t u d i e d : TTBP, t h e s t a b l e f r e e r a d i c a l s and t h e g e n e r a t e d r a d i c a l s . 3.11 (a) T r i - t - b u t y l Phenoxy The TTBP r a d i c a l was used as an i n s t r u m e n t a l s t a n d a r d . T h i s was p r e p a r e d from t h e p a r e n t phenol ( A l d r i c h ) o x i d i z e d by d r y P b 0 2 i n o u t g a s s e d m i n e r a l o i l . The r a d i c a l c o n c e n t r a t i o n r e a c h e d a maximum o f a b o u t 0.1 M a f t e r 4 days and t h e r e a f t e r decayed. T h i s m i x t u r e was q u i t e s t a b l e i n a i r \ 70 f o r s e v e r a l minutes and t h u s e a s i l y h a n d l e d . S o l u t i o n s o f v a r i o u s c o n c e n -t r a t i o n s were made up v o l u m e t r i c a l l y from t h e s e s t o c k s o l u t i o n s w i t h an " o i l p i p e t t e " and s t a n d a r d v o l u m e t r i c t e c h n i q u e s . The p r e p a r e d s o l u t i o n s were i m m e d i a t e l y f r o z e n i n l a r g e ENDOR sample tubes and o u t -g a s s e d by t h e freeze-pump-thaw t e c h n i q u e . C h l o r o f o r m ( F i s h e r ) , c a r b o n t e t r a c h l o r i d e ( F i s h e r ) and d e u t e r o c h l o r o f o r m (98% D, Merck) were s p e c t r a l o g r a d e , and used a f t e r d r y i n g o v e r 4 A g e l . For t h e hydrogen bond s t u d y , some r e a c t i o n m i x t u r e s were a l s o made d i r e c t l y i n c h l o r o f o r m o r c a r b o n t e t r a c h l o r i d e , but t h e s e were h i g h l y u n s t a b l e u n l e s s f r o z e n . The s p e c t r o m e t e r g i v e s c o u p l i n g c o n s t a n t s a c c u r a t e t o w i t h i n 30 kHz w i t h no p a r t i c u l a r s o p h i s t i c a t i o n . However, i n t h e c a s e o f TTBP, one o f t h e e f f e c t s was l e s s t h a n t h i s , so more c a r e was n e c e s s a r y . The mid p o i n t o f the d e r i v a t i v e c u r v e was o b t a i n e d a t as low an fm m o d u l a t i o n as p o s s i b l e (20 kHz) and s e v e r a l seconds were a l l o w e d f o r t h e system t o e q u i l i b r a t e a t t h i s p o i n t . Both l i n e s i n t h e ENDOR spe c t r u m were measured t w i c e w i t h i n minutes and a v e r a g e s o b t a i n e d . A S/N > 100 i s n e c e s s a r y f o r good r e s u l t s . T h i s p r o c e d u r e gave r e p r o d u c i b i l i t y i n a 5 MHz c o u p l i n g t o w i t h i n ± 4 kHz. 3.11 (b) S t a b l e F r e e R a d i c a l s DPPH samples were p r e p a r e d by d i s s o l v i n g c r y s t a l l i n e DPPH ( A l d r i c h Chemical Co., r e a g e n t grade) i n n-heptane and then d i s s o l v i n g t h i s s o l u t i o n o f . o u t g a s s e d m i n e r a l o i l . The samples were pumped on u n t i l most o f the n-heptane had e v a p o r a t e d , t h e n d e g a s s e d by t h e f r e e z e -71 thaw t e c h n i q u e and by l o w e r i n g t h e p r e s s u r e t o about 0.1 t o r r . The use o f n-heptane was not i m p o r t a n t , i t s i m p l y saved t i m e i n d i s s o l v i n g DPPH. Some samples were a l s o p r e p a r e d by d i s s o l v i n g DPPH d i r e c t l y i n m i n e r a l o i l and t h e y a l s o gave good ENDOR enhancements. ENDOR was a l s o o b t a i n e d on DPPH i n L i c r i s t a l phase IV, a l i q u i d c r y s t a l , by o u t g a s s i n g w i t h t h e same t e c h n i q u e . The phase IV was heat e d t o 120° t o dry ; i t , outgassed. and used w i t h o u t f u r t h e r p u r i f i c a t i o n . For ENDOR t h e c o n c e n t r a t i o n o f r a d i c a l was abo u t 1 0 ~ 2 M i n m i n e r a l o i l and was a d j u s t e d t o o b t a i n . a r e s o l v e d 5 - l i n e EPR spectrum a t 40°C b u t a broadened and u n r e s o l v e d s i n g l e l i n e a t room t e m p e r a t u r e . For ENDOR o f DPPH, t h e EPR must be as s t r o n g as p o s s i b l e , y e t s t i l l show microwave s a t u r a t i o n : t h e s e c o n d i t i o n s were found t o be q u i t e c r i t i c a l f o r o b t a i n i n g any ENDOR on t h i s compound. F o r a w e l 1 - r e s o l v e d EPR o f DPPH, t h e c o n c e n t r a t i o n was much l e s s , about 1 0 _ l + M i n n-heptane. These s o l u t i o n s must be o u t g a s s e d f o r s e v e r a l hours t o o b t a i n a m u l t i - l i n e spectrum w i t h optimum r e s o l u t i o n . P i c r y l - N - a m i n o c a r b a z y l (PAC) r a d i c a l s were o b t a i n e d i n pure c r y s t a l l i n e form, 'K and K L a b o r a t o r i e s ; BDPA was o b t a i n e d from A l d r i c h Chemical Company. BDPA d i s s o l v e s q u i t e e a s i l y i n warm m i n e r a l o i l , w h i l e PAC ENDOR samples were made by c a r r y i n g t h e d i s s o l v e d PAC i n w i t h n-heptane. F o r the EPR o f both t h e s e r a d i c a l s , s p e c t r a l grade d r i e d benzene and n-heptane were used. C o n c e n t r a t i o n s were a d j u s t e d as f o r DPPH t o o b t a i n optimum r e s o l u t i o n . With t h e narrow BDPA EPR sp e c t r u m , i t was n o t p o s s i b l e t o o b t a i n a c o m p l e t e l y r e s o l v e d EPR, t h e l i m i t i n g f a c t o r b e i n g t h e 100 kHz m o d u l a t i o n . 72 Both o f t h e s e samples g i v e a s l i g h t l y h i g h e r enhancement than DPPH, so t h a t a s t u d y o v e r a range o f t e m p e r a t u r e s c o u l d be made. In t h e s e c a s e s a low fm d e v i a t i o n (about 7 kHz) was employed t o r e v e a l t h e v e r y s m a l l d i f f e r e n c e s v i s i b l e i n t h e lower t e m p e r a t u r e s p e c t r a . 3.11 (c) G e n e r a t e d R a d i c a l s The r e a c t i o n between DPPH and TTBP ( o r i t s i s o - p r o p y l a n a l o g u e ) 39 40 has been s t u d i e d by Ayscough and R u s s e l l . 5 In t h e absence o f oxygen, t h e s e s p e c i e s b l e a c h DPPH t o form a r a d i c a l s a n d .the DPPH r a d i c a l i s d e s t r o y e d . F u r t h e r m o r e , t he DPPH does n o t r e a c t w i t h t h e new r a d i c a l , and no s o l v e n t -DPPH complexes were d e t e c t e d , t o the l i m i t o f the EPR i n s t r u m e n t u s e d . T h i s method was thus t h o u g h t t o be a p p l i c a b l e t o o t h e r s y s t e m s , i f t h e r e l a t i v e c o n c e n t r a t i o n s c o u l d be c o n t r o l l e d . I f t h e r a d i c a l p a r e n t 40 R c o n t a i n s an a b s t r a c t a b l e p r o t o n t h e r e a c t i o n can be r e p r e s e n t e d by R-H + DPPH- v R- + DPPH-H [53] 40 The h y d r a z i n e w i l l n o t r e a c t f u r t h e r . T h i s a b s t r a c t i o n t e c h n i q u e was p e r f e c t e d f o r s e v e r a l o f t h e t h i a z i n e and o x a z i n e compounds which had a b s t r a c t a b l e p r o t o n s . A l l o f t h e s p e c i e s t r i e d b l e a c h e d DPPH w i t h i n 15 m i n u t e s , b ut s e v e r a l gave o n l y d i a m a g n e t i c p r o d u c t s and no EPR s p e c t r a . P h e n o x a z i n e ( f t l d r i c h ) , p h e n o t h i a z i n e ( F i s h e r ) and c h l o r o -p h e n o t h i a z i n e ^ I d r i c h ) gave good ENDOR by t h i s t e c h n i q u e . 73 A s a t u r a t e d s o l u t i o n ( 1 0 ~ 2 M) o f t h e p a r e n t was p r e p a r e d i n o u t g a s s e d m i n e r a l o i l and f r o z e n i n a sample t u b e . A v e r y c o n c e n t r a t e d s o l u t i o n o f DPPH i n n-heptane was added d r o p w i s e t o t h e f r o z e n s o l u t i o n . The added n-heptane-DPPH s o l u t i o n . was o u t g a s s e d by freeze-pump-thaw t e c h n i q u e s and then t he m i n e r a l o i l s o l u t i o n would be m e l t e d g e n t l y . A f t e r m i x i n g and b l e a c h i n g o f t h e DPPH, the e x c e s s n-heptane was s t r i p p e d o f f by pumping. EPR samples were p r e p a r e d i n a s i m i l a r way w i t h o u t min-e r a l o i l , and t h o r o u g h l y o u t g a s s e d n-heptane o n l y . Any samples which gave an i n i t i a l EPR spectrum o f DPPH were d i s c a r d e d as i n c o m p l e t e r e a c t i o n systems. P h e n y l a r s a z i n e and i m i n o d i b e n z y l b l e a c h e d DPPH q u i t e r a p i d l y but gave no EPR: f u r t h e r r e a c t i o n s must have been t a k i n g p l a c e . Some o f th e d y e s , such as Medola B l u e gave no b l e a c h i n g . These a r e presumably so d i s t o r t e d t h a t s t e r i c h i n d r a n c e b l o c k s t h e DPPH r e a c t i o n . As l o n g as t h e y were t h o r o u g h l y o u t g a s s e d , r a d i c a l s p r e p a r e d t h i s way were s t a b l e f o r s e v e r a l hours i n n-heptane and up t o 5 hours i n m i n e r a l o i l . The l i m i t i n g f a c t o r i s not t h e r e a c t a b i l i t y w i t h DPPH, but th e low s o l u b i l i t y o f the p a r e n t i n m i n e r a l o i l . However, S/N g r e a t e r than 50 was o b t a i n e d on the ENDOR o f most o f t h e s e compounds. R a d i c a l s p r e p a r e d t h i s way gave a microwave s a t u r a t i o n around 20 mw i n m i n e r a l o i l , so t h a t low n o i s e ENDOR c o u l d u s u a l l y be o b t a i n e d . A l l o f t h e s e s p e c i e s have v e r y wide ENDOR s p e c t r a . The s i g n a l g e n e r a t o r used has a band s w i t c h - o v e r a t 10 MHz w i t h a d i f f e r e n t fm d e v i a t i o n above and below t h i s p o i n t . T h i s a c c o u n t s f o r t h e i n t e n s i t y a n o m a l i e s o b s e r v e d i n t h e low f i e l d l i n e s o f t h e s e samples. 74 CHAPTER FOUR RESULTS AND DISCUSSIONS I n t r o d u c t i o n T h r e e d i s t i n c t s t u d i e s were u n d e r t a k e n w i t h t h e completed s p e c t r o m e t e r . The f i r s t was a d e t a i l e d s t u d y o f hydrogen bond-complex f o r m a t i o n i n s o l u t i o n s o f TTBP r a d i c a l . S e c o n d l y , a s e r i e s o f s t a b l e f r e e r a d i c a l s , DPPH, BDPA and PAC was s t u d i e d i n an attempt t o u n r a v e l t h e c o n t r o v e r s y s u r r o u n d i n g t h e i r v e r y complex h i g h - r e s o l u t i o n EPR s p e c t r a . F i n a l l y , a s e r i e s o f g e n e r a t e d r a d i c a l s o f b i o l o g i c a l i n t e r e s t was- s t u d i e d i n o r d e r t o expand the ENDOR t e c h n i q u e t o l e s s s p e c i a l i z e d m o l e c u l e s . 75 4.1 The Phenoxy R a d i c a l and Hydrogen Bonding The t e r t - t - b u t y l Phenoxy (TTBP) r a d i c a l whose s t r u c t u r e i s shown i n F i g u r e 9 has a l r e a d y been s t u d i e d e x t e n s i v e l y w i t h ENDOR by A l l e n d o e r f e r and M a k i J ° From a phe n o m e n o l o g i c a l v i e w p o i n t , t h e y l o o k e d a t t h e TTBP ENDOR enhancement as a f u n c t i o n o f microwave power, r f power, v i s c o s i t y , t e m p e r a t u r e and m o d u l a t i o n f r e q u e n c y . I t was t h i s d e t a i l e d work which r e s u l t e d i n the c h o i c e o f TTBP as t h e f i r s t r a d i c a l t o be s t u d i e d on t h e completed s p e c t r o m e t e r . TTBP meets many o f the r e q u i r e m e n t s f o r an ENDOR s t a n d a r d . I t i s ea s y t o p r e p a r e ; i t s a t u r a t e s a t low microwave power; i t has a narrow EPR spectrum which can be modulated o v e r i t s t o t a l w i d t h (4 G a u s s ) . The ENDOR l i n e s a r e w e l l s e p a r a t e d ( s e e F i g 9) and a l l o f the l i n e s a r e q u i t e v i s i b l e even w i t h t h e r e d u c t i o n from t h e T 2 c o r r e c t i o n f o r m u l a ( e q u a t i o n [30]) because o f the l a r g e number o f p r o t o n s r e s p o n s i b l e f o r t h e c e n t r a l l i n e s . An ENDOR enhancement s t a n d a r d would have a l l o f t h e s e p r o p e r -t i e s ; a s p e c i f i c sample w i t h measured enhancement would t h e n be used t o d e t e c t i n s t r u m e n t a l d e t e r i o r a t i o n . U n f o r t u n a t e l y , samples o f TTBP kept f o r s e v e r a l weeks decay t o a second r a d i c a l , whose ENDOR i s shown i n F i g 10. A l l o f t h e v a r i o u s samples o f TTBP e v e n t u a l l y gave a spectrum s i m i l a r t o 10 ( c ) , a l t h o u g h some'samples took l o n g e r t h a n o t h e r s t o r e a c h t h i s s t a g e . T h i s d e c a y makes TTBP u n a c c e p t a b l e as a l o n g term s t a n d a r d . A t h e r t o n e t a l ^ " have o b s e r v e d s i m i l a r s e c o n d a r y r a d i c a l s i n a s e r i e s o f s u b s t i t u t e d phenoxy r a d i c a l s . In t h i s work t h e y d i s c u s s F i g u r e 9. T y p i c a l ENDOR o f t e r t - t - b u t y l phenoxy i n c a r b o n t e t r a c h l o r i d e (5%) and m i n e r a l o i l a t 20°C. The f r e e p r o t o n f r e q u e n c y and t h e a s s i g n m e n t s o f t h e p r o t o n s a r e shown. F i g u r e 10. ENDOR o f TTBP i n m i n e r a l o i l (100%) a f t e r a g i n g , showing t h e a p p e a r a n c e o f l i n e s from the s e c o n d a r y o x i d a t i o n s p e c i e s . 78 c o m p l e t e l y t h e p o s s i b l e s e c o n d a r y s p e c i e s i n the o x i d a t i o n o f t h e v a r i o u s phenoxy compounds. These i n c l u d e a r y l o x y d e r i v a t i v e s , dimers o f v a r i o u s t y p e s , and ketone phenoxy d e r i v a t i v e s . They a r e un a b l e t o d e t e r m i n e e x a c t l y t h e n a t u r e o f t h e s e c o n d a r y p r o d u c t i n TTBP. In t h i s c a s e t h e ENDOR i s n o t v e r y c o n c l u s i v e , a l t h o u g h t h e new l i n e s a t 12 and 15 MHz do s u g g e s t a n o t h e r meta r i n g p r o t o n , from t h e i r i n t e n s i t y and measured c o u p l i n g . T h i s would s u g g e s t some d i m e r i z a -t i o n t o g i v e a second s p e c i e s w i t h 2 phenyl r i n g s and one u n p a i r e d e l e c t r o n : ( T T B P ) 2 . N e i t h e r t h e EPR nor the-ENDOR-induced-EPR 5 r e v e a l a n y t h i n g e l s e about t h i s s p e c i e s : both spectrum show t h e t h r e e l i n e s , s i m i l a r t o TTBP, but w i t h s m a l l e r c o u p l i n g . . The p o s s i b l e c h e m i c a l p a t h -ways i n t h i s system a r e c o m p l i c a t e d . F u r t h e r m o r e , t h e TTBP d i d not l i v e f o r l o n g enough i n t h i n s o l v e n t s such as n-heptane t o g i v e t h e s e c o n d a r y s p e c i e s a chance t o app e a r . More d e t a i l e d s t u d i e s on TTBP s o l u t i o n s do r e v e a l t h e e x i s t e n c e o f a weak hydrogen bond between TTBP and p r o t o n - d o n a t i n g s o l v e n t s . A d e t a i l e d s t u d y o f the dynamics o f t h i s bond u s i n g t h e t h e o r y o f S e c t i o n 2.8 i n d i c a t e s t h a t ENDOR can b e s u c c e s s f u l l y used a a d i a g n o s t i c t o o l f o r s h o r t - l i v e d weak complexes which might n o t be d e t e c t a b l e w i t h e i t h e r EPR o r NMR. TTBP has been i n v e s t i g a t e d i n s o l u t i o n s o f CHC1 3, CDC1 3, C C l ^ 41 and n-heptane. P r e v i o u s a t t e m p t s by Buchachenko e t a l t o d e t e c t h y d r o g e n - b o n d i n g i n t h i s system by u s i n g o p t i c a l a b s o r p t i o n were u n s u c c e s s -42 f u l . L a t e r t h e s e a u t h o r s a l s o used EPR t o s t u d y h y d r o g e n - b o n d i n g i n s o l u t i o n s o f the ( a n a l o g o u s ) C o p p i n g e r ' s r a d i c a l . They have c o n c l u d e d t h a t 79 l i k e TTBP, C o p p i n g e r ' s r a d i c a l e i t h e r does n o t "form hydrogen bonds w i t h p r o t o n - d o n a t i n g s o l v e n t s , o r t h a t t h e e q u i l i b r i u m i s s h i f t e d towards t h e non-hydrogen-bonded s p e c i e s . More r e c e n t l y u s i n g EPR, 43 Mukai e t a l a l s o c o n c l u d e d t h a t hydrogen bonds were not formed i n s o l u t i o n s o f TTBP i n s e v e r a l p r o t o n d o n a t i n g s o l v e n t s . The h i g h e r r e s o l u t i o n p o s s i b l e w i t h t h e ENDOR t e c h n i q u e was used t o measure a f o r m a t i o n c o n s t a n t , and hence t o e s t i m a t e t h e enthalpy of formation f o r t h e complex f o r m a t i o n . F u r t h e r m o r e , t h e e f f e c t o f t h e complex f o r m a t i o n i s o b s e r v e d f r o m - t h e r e c e p t o r s i d e o f t h e m o l e c u l e , r a t h e r than on t h e a c c e p t o r s i d e , as i s done i n the NMR s t u d i e s o f hydrogen bonding i n more s t r o n g l y hydrogen-bonded complexes. In t h i s r e s p e c t t h e p r e s e n t s t u d i e s a r e b e l i e v e d t o be i m p o r t a n t f o r e x t e n d i n g t h e p o s s i b i l i t i e s f o r s t u d y i n g weaker and f a s t e r c o m p l e x a t i o n s i n s o l u t i o n s . The ENDOR method complements even t h e r e s u l t s o b t a i n e d t h r o u g h t h e Dynamic N u c l e a r P o l a r i -z a t i o n (DNP) t e c h n i q u e , because t h e ti m e s c a l e o f ENDOR i s h i g h e r than t h a t o f DNP, even though s e n s i t i v i t y and r e s o l u t i o n o f t h e two t e c h n i q u e s may be n e a r l y t h e same. In a d d i t i o n , t h e hydrogen bond s t u d i e d here i s an 0 H-C bond which i s a r a t h e r unusual hydrogen bond. Such hydrogen bonds have been r e c e n t l y p o s t u l a t e d ^ t o a c c o u n t f o r v a r i o u s c o n f o r m a t i o n s o f s e v e r a l p e p t i d e u n i t s i n b i o l o g i c a l s y s t e m s . Some e x p e r i m e n t a l d a t a on t h e forma-t i o n o f t h e s e bonds ( i n somewhat d i f f e r e n t s y s t e m s ) h a v e been r e c e n t l y o b t a i n e d t h r o u g h NMR 4 5' 4 6 and E P R 4 7 t e c h n i q u e s . These s t u d i e s used n i t r o x i d e r a d i c a l s where t h e hydrogen bonds formed a r e c o m p a r a t i v e l y e a s i e r t o d e t e c t because t h e oxygen o f the n i t r o x y l group i s n o t h i n d e r e d b y t h e t - b u t y l groups and can form i n t e r m o l e c u l a r hydrogen bonds w i t h v a r i o u s p r o t o n -80 d o n a t i n g s o l v e n t s . In t h e c a s e o f t h e TTBP-type r a d i c a l s t h e oxygen o f th e phenoxy! group i s e x p e c t e d t o be s h i e l d e d by t h e t - b u t y l g r o u p s . The hydrogen-bonds formed i n t h e TTBP-type r a d i c a l s a r e t h e r e f o r e c o m p a r a t i v e l y much weaker. T h i s i s p r o b a b l y why t h e y have not been d e t e c t e d e a r l i e r . A m o l e c u l a r - o r b i t a l s t u d y o f t h e f o r m a t i o n o f C-H 0 bonds has a l s o 48 a p p e a r e d r e c e n t l y . The r e s u l t s o b t a i n e d t h r o u g h ENDOR i n t h e p r e s e n t work w i l l be compared w i t h t h e above mentioned s t u d i e s . The s e l e c t i o n o f a t e m p e r a t u r e a t which t o s t u d y t h i s system r e q u i r e d some p r e l i m i n a r y e x p e r i m e n t s . In pure m i n e r a l o i l , ENDOR o f TTBP i s v i s i b l e o v e r a 120°C range. As s o l v e n t s a r e added, t h i s "ENDOR window" narrows c o n s i d e r a b l y , u n t i l a t about 70% CHC1 3 the narrow window, t h e m i c r o -wave c o u p l i n g problem, and t h e ( n e c e s s a r i l y ) s m a l l e r samples a l l combine to g i v e no ENDOR a t t h e r a d i c a l c o n c e n t r a t i o n s used. With a more t y p i c a l c on-c e n t r a t i o n , say 25%, t h e ends o f the window a r e w e l l c h a r a c t e r i z e d . A t th e l o w e r l i m i t the m i x t u r e f r e e z e s and t h e g l a s s y s p e c t r a a r e v e r y d i s -t o r t e d and depend c r i t i c a l l y upon t h e magnetic f i e l d s t r e n g t h . A t t h e h i g h -t e m p e r a t u r e l i m i t , t h e ENDOR l i n e w i d t h goes f r o m 70 kHz t o 600 kHz, making i t d i f f i c u l t t o a c c u r a t e l y measure and f o l l o w s m a l l changes. The l a r g e change i n T 2 f r o m e q u a t i o n [30] a l s o d i s t o r t s t h e l i n e i n t e n s i t i e s , and t h e S/N f a l l s . With a l l t h e s e c o n s i d e r a t i o n s i n mind, a c e n t r a l t e m p e r a t u r e o f -30°C was c h o s e n . Pure m i n e r a l o i l i s a l m o s t f r o z e n a t t h i s "low" t e m p e r a t u r e , and 50% CHC1,3 i s showing s i g n s o f h i g h t e m p e r a t u r e d i s t o r t i o n a t t h i s " h i g h " t e m p e r a t u r e . S t u d i e s done above and below t h i s t e m p e r a t u r e n e c e s s a r i l y have fewer p o i n t s . F i g u r e 9 shows a t y p i c a l ENDOR spectrum o f TTBP i n a mixed s o l v e n t system w i t h 5% C C 1 4 i n m i n e r a l o i l . The spectrum r e s e m b l e s t h a t 81 o b s e r v e d i n m i n e r a l o i l . The a s s i g n m e n t o f t h e l i n e s t o the v a r i o u s p r o t o n s i s as shown i n t h e diagram. The l i n e s near 11 and 16 MHz, due t o t h e meta r i n g p r o t o n s , were f o u n d t o be the most s e n s i t i v e t o hydro-gen bond complex f o r m a t i o n . Under i d e n t i c a l c o n d i t i o n s , t h e EPR shows t h e s e l i n e s , b u t t h e a c c u r a t e v a l u e o f the c o u p l i n g i s masked by t h e o t h e r t - b u t y l l i n e s . F u r t h e r d i s c u s s i o n c o n c e r n s t h e v a r i a t i o n i n t h e s e meta-proton l i n e p o s i t i o n s . Both t h e l i n e p o s i t i o n s and l i n e - w i d t h s o f the meta-proton ENDOR s i g n a l s showed d e f i n i t e changes w i t h added s o l v e n t s and w i t h t e m p e r a t u r e v a r i a t i o n . The f r a c t i o n a l change w i t h t e m p e r a t u r e was, however, found t o be s m a l l e r than t h a t w i t h c h a n g i n g s o l v e n t s . T h i s can be seen from F i g 11 where t h e v a r i a t i o n w i t h t e m p e r a t u r e o f t h i s c o u p l i n g w i t h some t y p i c a l s o l v e n t s i s shown. The g e n e r a l t r e n d f o r t h e te m p e r a t u r e v a r i a t i o n o f a l l samples i s s i m i l a r t o t h a t f o r n-heptane, s t u d i e d e a r l i e r by A l l e n d o e r f e r and Maki J ° I t may be no t e d from F i g 11 t h a t t h e range o f t e m p e r a t u r e s o v e r which ENDOR c o u l d be o b s e r v e d i n . t h e s e e x p e r i m e n t s ( d e f i n e d above as the ENDOR 'window'), becomes narrower w i t h i n c r e a s i n g c o n c e n t r a t i o n o f p r o t o n - d o n a t i n g s o l v e n t s . The v a r i a t i o n o f t h i s d a t a a t the s e l e c t e d t e m p e r a t u r e o f -30° as a f u n c t i o n o f concentration(m / J t ) i s shown i n F i g 12. T h e r e i s a c l e a r d e c r e a s e o f about 10% i n the r i n g - p r o t o n c o u p l i n g w i t h CHC1 3 and v e r y l i t t l e change w i t h added CC1^. T h i s i s t h e e f f e c t o f the complex forma-t i o n on the s p i n d e n s i t y a t t h e meta p r o t o n s . T h a t t h i s e f f e c t i s due to hydrogen bond f o r m a t i o n was shown by subsequent e x p e r i m e n t s . The s m a l l d e c r e a s e i n t h i s CCI^ c o u p l i n g i s presumably caused by a d i f f e r e n c e i n v i s c o s i t y between t h e samples. In p r i n c i p l e , t h e s e c o u p l i n g s 82 F i g u r e 11, Te m p e r a t u r e v a r i a t i o n o f m e t a - p r o t o n c o u p l i n g c o n s t a n t i n TTBP. A few o f t h e s o l v e n t c o n c e n t r a t i o n s s t u d i e d a r e shown. 83 • C C l 4 0 C H C I 3 ACDCI3 •frozen samples 1 1 1 1 1 1 2 3 4 5 [Slm/l 12. C o n c e n t r a t i o n dependence o f TTBP meta p r o t o n c o u p l i n g c o n s t a n t a t -30°C w i t h added CC1,,, CHC1 3 and CDC1 3. 84 s h o u l d be measured a t c o n s t a n t T/n i n s t e a d o f c o n s t a n t T; however, the v i s c o s i t y d a t a f o r such mixed systems i s not a v a i l a b l e . The d i f f e r e n c e i n c o u p l i n g c a u s e d by i n c l u s i o n o f t h i s e r r o r i s o n l y about 4%. Recent work by 4Q Kecki and C i e s l a k i n d i c a t e s t h a t c a r b o n t e t r a c h l o r i d e i s n o t . a s - " i n e r t " a s o l v e n t as n-heptane, "and t h a t some v e r y weak hydrogen bond complex f o r m a t i o n between c a r b o n t e t r a c h l o r i d e and methanol does o c c u r . In t h i s c a s e , t h i s (4%) d e c r e a s e i n the c o u p l i n g w i t h added c a r b o n t e t r a c h l o r i d e might be e v i d e n c e o f t h i s t y p e o f weak complex f o r m a t i o n . In o r d e r t o e s t a b l i s h t h a t t h i s was a complex f o r m a t i o n c o n s t a n t , t h i s d a t a and measurements a t o t h e r t e m p e r a t u r e s were t r e a t e d by t h e S c a t c h a r d p l o t u s i n g e q u a t i o n [ 4 9 ] : <5/[CHCl3] v e r s u s 6. P o i n t s l e s s than 0.2(6 - 6 ) were r e j e c t e d when 6Q was o b t a i n e d from t h i s t r e a t m e n t . The r e s u l t s a t -30°C a r e shown i n F i g 13. The arrowheads show the s a t u r a t i o n l i m i t s o f S - 0.2and S = 0 . 8 . I t i s i m p o s s i b l e t o o b t a i n a s a t u r a t i o n o f more than S = 0.8 s i n c e even 100% CHC1 3 i s o n l y 12.4 M. F u r t h e r m o r e , no ENDOR was o b s e r v e d above 6 M CHC1 3 where S = 0.45. N e v e r t h e l e s s , a l l 28 the p o i n t s used a r e s t i l l i n D e r a n l e a u ' s a c c e p t e d r a n g e . The d a t a were l e a s t - s q u a r e s - f i t t e d t o a s t r a i g h t l i n e and the s l o p e o f t h i s p l o t g i v e s -3 0° K f = 0.27 ± 0.02. T h i s a g r e e s w e l l w i t h f o r m a t i o n c o n s t a n t s f o r hydrogen 45 bonds s t u d i e d by NMR where K v a l u e s f o r c h l o r o f o r m were about 0.2 a t room t e m p e r a t u r e and h i g h e r f o r o t h e r s o l v e n t s . T h i s s m a l l v a l u e shows t h e p r e s e n c e o f a f a i r l y weak hydrogen bond. I t s h o u l d be n o t e d t h a t t h e v a l u e o f t h i s f o r m a t i o n c o n s t a n t i s i n d e p e n d e n t on the p o i n t o f o b s e r v a t i o n . A l t h o u g h t h i s i s o n l y a s m a l l change i n s p i n d e n s i t y on a p r o t o n 3 carbons away from t h e a c t i v e s i t e o f t h e bond f o r m a t i o n , t h i s t r e a t m e n t w i l l s t i l l g i v e c o r r e c t K f v a l u e s . However, i t i s d o u b t f u l i f EPR a l o n e c o u l d have 85 .00-0.1 0.2 s ( M H z ) 0.3 0.4 F i g u r e 13. S c a t c h a r d P l o t o f meta p r o t o n c o u p l i n g c o n s t a n t a t TTBP a t -30°C i n CHC1 3. The arrows i n d i c a t e t h e 6 l i m i t s o f 0.2 and 0.8. 86 d e t e c t e d t h i s s m a l l change. The e x p e r i m e n t a l l y u n o b t a i n a b l e 6Q i s 0.435 from t h e i n t e r c e p t o f t h e S c a t c h a r d p l o t , and t h i s was used t o f i n d t h e 0.2 and 0.8 S l i m i t s . H i g h e r and lower t e m p e r a t u r e s gave K's as shown i n T a b l e 4, but because o f the n a r r o w i n g o f t h e ENDOR window, t h e s e o t h e r t e m p e r a t u r e s have fewer p o i n t s and a r e l e s s r e l i a b l e . A v a n ' t H o f f p l o t 5 0 o f l o g K a g a i n s t T " 1 g i v e s an e n t h a l p y o f forma-t i o n o f AH = 3.5 ±' 1 k c a l . T h i s a l s o a g r e e s w i t h t h e NMR d a t a 4 5 on r e l a t e d s ystems. TABLE 4 The ENDOR d e t e r m i n e d f o r m a t i o n c o n s t a n t s and e n t h a l p y o f f o r m a t i o n o f  hydrogen bonds i n CHC1 3 TTBP s o l u t i o n s . -T(K) K f o r m ^ / m ^ A H ( k c a l / m o l e ) 253 .21 ± .03 ) 243 .26 ± .02 -3.5 ± 1 233 .45 ± .03 A s t r o n g s u p p o r t f o r the p o s t u l a t e t h a t t h e o b s e r v e d e f f e c t i s due t o hydrogen bond f o r m a t i o n was o b t a i n e d by d u p l i c a t i o n o f t h e CHC1 3 samples w i t h 90% CDC1 3. The d i f f e r e n c e i n the c o u p l i n g s as a f u n c t i o n o f [S ] i s shown i n F i g 14. A l t h o u g h t h i s d i f f e r e n c e i s s m a l l , t h e t r e n d w i t h [ S ] , t h e r e p r o d u c i b i l i t y o f s e v e r a l samples and a c c u r a t e e s t i m a t e o f the e r r o r s shows t h i s i s o t o p e ^ . s h i f t t o be r e a l . Care must be t a k e n t o measure such a sma l l s h i f t , even w i t h ENDOR, as d i s c u s s e d i n s e c t i o n 3.11 ( a ) . % S $ ) ( C H G I 3 o r CDCI3) F i g u r e 14. The i s o t o p e e f f e c t on t h e hydrogen bond: c o n c e n t r a t i o n depen-dence o f t h e d i f f e r e n c e i n meta D r o t o n , c o u p l i n g i n TTBP between added CHC!3 and C D C 1 3 . 88 The o b s e r v e d s h i f t i n d i c a t e s than an e f f e c t s i m i l a r t o t h e 51 Ubbelohde E f f e c t f o r 0-H 0 bonds a l s o o c c u r s i n C-H 0 bonds. Because o f the z e r o - p o i n t m o t i o n , t h e 0-D bond l e n g t h i s g e n e r a l l y s h o r t e r than t h e c o r r e s p o n d i n g 0-H bond, hence 0 D-0 bonds a r e gener-a l l y l a r g e r t h a n t h e c o r r e s p o n d i n g 0 H-0 bonds (Ubbelhode E f f e c t ) . The p r e s e n t o b s e r v a t i o n t h a t the change i n t h e c o u p l i n g c o n s t a n t i s s m a l l e r f o r CDC1 3 i n d i c a t e s t h a t 0 D-C bond i s l o n g e r than t h e c o r r e s p o n d i n g 0 H-C bond. T h i s magnitude o f the e f f e c t (about 5%) i s - o f AC the same o r d e r o f magnitude as f o r t h e 0-H 0 bonds, o b s e r v e d t h r o u g h NMR. The s u c c e s s o f t h e p r e s e n t work shows t h a t t h e ENDOR t e c h n i q u e can be used t o s t u d y weak complex f o r m a t i o n i n s o l u t i o n s . A l t h o u g h a p p l i e d t o hydrogen bonding i n t h i s work t he p r o c e d u r e o u t l i n e d here s h o u l d be g e n e r a l l y a p p l i c a b l e t o any weak complex f o r m a t i o n . The h i g h e r r e s o l u t i o n and time, s c a l e o f t h e ENDOR t e c h n i q u e might make p o s s i b l e t h e s t u d y o f p r o c e s s e s such as t h e s e t h a t a r e not d e t e c t e d by o t h e r t e c h n i q u e s . 4.2 a , g'-Dipheny1-g-Picry1 H y d r a z y l (DPPH) The s t a b l e f r e e r a d i c a l a , a ' - D i p h e n y l - 3 - P i c r y l H y d r a z y l (DPPH), whose m o l e c u l a r s t r u c t u r e and numbering system a r e shown i n F i g 15, i s one o f t h e most commonly used r e a g e n t s i n f r e e - r a d i c a l c h e m i s t r y and i n 52 s o l u t i o n i t shows v a r y i n g m a g n e t i c p r o p e r t i e s depending upon t he s o l v e n t . A l l t h e s e p r o p e r t i e s i n d i c a t e t h a t t h e u n p a i r e d e l e c t r o n i s e x t e n s i v e l y d e l o c a l i z e d o v e r t h e e n t i r e m o l e c u l a r s t r u c t u r e . C o n s i d e r a b l e e f f o r t has t h e r e f o r e been d e v o t e d i n at t e m p t s t o e s t i m a t e t h i s u n p a i r e d e l e c t r o n s p i n D P P H 3 2 > \ c o u P ' ' n < 3 s ' n G a u s s ) + £ 7 * 4 V2 .O N C ^ ( a 7 ) + . 6 7 < v 9 (79) (9.7) •2.0 g u r e 15. a , a ' - D i p h e n y l - 3 - P i c r y l h y d r a z y l r a d i c a l s t r u c t u r e , showing numbering sys t e m and c o u p l i n g s o b t a i n e d ( G ) . 90 d e n s i t y d i s t r i b u t i o n . DPPH has been t he s u b j e c t o f many EPR s t u d i e s . 5 3 - 6 8 The e a r l i e r s t u d i e s were done on s o l i d DPPH, the spectrum o f which c o n s i s t s o f o n l y one l i n e . 5 ^ T h i s s h a r p s i g n a l a r i s e s from t h e extreme e x c h a n g e - n a r r o w i n g 6 9 which o b l i t e r a t e s a l l h y p e r f i n e s t r u c t u r e . In d i l u t e d s o l i d s o l u t i o n s h y p e r f i n e s t r u c t u r e from t h e c e n t r a l N^ and N^ n i t r o g e n s has been r e s o l v e d . Deal and Koski , 5 6 u s i n g computer s i m u l a t i o n t e c h n i q u e s , f i r s t f o u n d t h a t t h e h y p e r f i n e i n t e r a c t i o n o f t h e u n p a i r e d e l e c t r o n was d i f f e r e n t f o r and N , the r a t i o o f t h e c o u p l i n g c o n s t a n t s b e i n g n e a r l y 0.82. U s i n g N 1 5 CO s u b s t i t u t i o n , Chen e t a l . " showed t h a t t h e l a r g e r n i t r o g e n c o u p l i n g i s from t h e h y d r a z y l (e) n i t r o g e n . S e v e r a l o t h e r EPR s t u d i e s showing s o l v e n t -dependent n i t r o g e n h y p e r f i n e c o u p l i n g s have a l s o been r e p o r t e d , 6 ^ 0 but t h e s e s t u d i e s d i d n o t y i e l d i n f o r m a t i o n on the r i n g - d e l o c a l i z a t i o n o f t h e 68 u n p a i r e d e l e c t r o n . In 1960, Deguchi r e p o r t e d t h e o b s e r v a t i o n o f p r o t o n h y p e r f i n e s t r u c t u r e i n the EPR s p e c t r a o f c a r e f u l l y degassed s o l u t i o n s o f DPPH. T h i s showed t h a t t h e e l e c t r o n was d e l o c a l i z e d o v e r t h e whole m o l e c u l e , but t h e s p e c t r a v/ere t o o complex f o r a n a l y s i s . Q u a l i t a t i v e e v i d e n c e f o r s p i n d e n s i t y d e l o c a l i z a t i o n has been o b t a i n e d . t h r o u g h t h e e f f e c t s o f r i n g s u b s t i t u t i o n on t h e o p t i c a l a b s o r p t i o n s p e c t r a o f the p a r e n t compound.^ NMR s t u d i e s have shed f u r t h e r l i g h t on p r o t o n h y p e r f i n e i n t e r -a c t i o n s i n t h i s r a d i c a l . The f i r s t q u a n t i t a t i v e measurements o f p r o t o n h y p e r f i n e c o u p l i n g s were o b t a i n e d t h r o u g h K n i g h t - s h i f t measurements i n 72 73 th e s o l i d phase. ' Because o f t h e r a t h e r l a r g e a n i s o t r o p y o f the h y p e r f i n e i n t e r a c t i o n , t h e r e s o l u t i o n was n o t good enough t o p e r m i t t h e d e t e r m i n a t i o n o f a l l o f the i n t e r a c t i o n c o n s t a n t s . 91 Hyde, Sneed and R i s t have used t he t e c h n i q u e o f E l e c t r o n -E l e c t r o n Double-Resonance ( E L D O R ) 7 0 t o s t u d y s o l u t i o n s o f DPPH and o b t a i n e d p r e c i s e v a l u e s f o r t h e h y p e r f i n e c o u p l i n g s o f t h e main n i t r o -gens, N^ and N . The ELDOR t e c h n i q u e d i d n o t g i v e any i n f o r m a t i o n about t h e s p i n d e n s i t y d i s t r i b u t i o n t h r o u g h o u t t h e r e s t o f t h e m o l e c u l e . 13 Hyde has alsoi:,mentioned u n s u c c e s s f u l a t t e m p t s t o o b s e r v e t h e ENDOR spectrum o f DPPH i n t h e l i q u i d phase. He t e n t a t i v e l y a t t r i b u t e d t h e f a i l u r e t o l a r g e a n i s o t r o p y i n t h e e l e c t r o n i c g v a l u e . On the t h e o r e t i c a l s i d e , o n l y a p p r o x i m a t e c a l c u l a t i o n s o f s p i n d e n s i t y d i s t r i b u t i o n had been a t t e m p t e d . The f i r s t d e t a i l e d work was 72 by Gutowsky e t a l . These a u t h o r s used a v a l e n c e - b o n d a p p r o x i m a t i o n t o c a l c u l a t e t h e u n p a i r e d e l e c t r o n s p i n d e n s i t y d i s t r i b u t i o n o v e r t h e whole m o l e c u l e . They o b t a i n e d a c c u r a t e v a l u e s f o r the s p i n d e n s i t i e s on t h e c e n t r a l n i t r o g e n atoms, N , and N„ but t h e i r e s t i m a t e s f o r the s p i n d e n s i t i e s on the r i n g p r o t o n s d i d not a g r e e w i t h v a l u e s e s t i m a t e d 72 73 74 from t h e NMR r e s u l t s . ' D e t a i l e d c a l c u l a t i o n s by W a l t e r a l s o y i e l d e d 67 75 p r e c i s e v a l u e s f o r s p i n d e n s i t i e s on N and N c . Gubanov e t a l . 5 have Cl p p r e s e n t e d r e s u l t s o f u n r e s t r i c t e d H a r t r e e - F o c k c a l c u l a t i o n s on the DPPH r a d i c a l . T h e o r e t i c a l c a l c u l a t i o n s a r e h e l p f u l i n a s s i g n i n g t h e o b s e r v e d ENDOR t r a n s i t i o n s , but f u r t h e r r e f i n e m e n t s a r e n e c e s s a r y t o g i v e s a t i s -f a c t o r y agreement w i t h t h e o b s e r v e d h y p e r f i n e c o u p l i n g s o b t a i n e d f r o m ENDOR. The DPPH ENDOR was o b t a i n e d i n o u t g a s s e d m i n e r a l o i l a t s l i g h t l y above room t e m p e r a t u r e . F i g u r e 16 shows t h e EPR o f t h i s sample and t h e ENDOR r e c o r d e d a t 30°C. I t c o n s i s t s o f 6 p a i r s o f l i n e s s y m m e t r i c a l l y p o s i t i o n e d around t h e f r e e p r o t o n f r e q u e n c y v H . The spectrum can be F i g u r e 16. (a) EPR o f DPPH sample i n m i n e r a l o i l used f o r ENDOR and (b) ENDOR s p e c t r u m o f same sample o f DPPH a t room t e m p e r a t u r e . 93 a n a l y s e d u s i n g t h e s p i n H a m i l t o n i a n o f e q u a t i o n [ I ] . F o r a l l s e v e n t e e n p a r a m a g n e t i c n u c l e i i n DPPH t h i s becomes: 36 = geeH-s +• .^(ajs.i'j - gH6HH-i") 1 2 : a ? S ^ - g H 3 H . I ? ) [54] The p r o t o n c o u p l i n g c o n s t a n t s o b t a i n e d from t h e ENDOR s p e c t r a a r e g i v e n i n T a b l e 5. The a s s i g n m e n t o f t h e s e c o u p l i n g s i s based upon NMR, ELDOR and t h e o r e t i c a l p r e d i c t i o n s , as w e l l as t h e i n t e n s i t i e s i n the ENDOR spectrum. ENDOR o f DPPH was a l s o o b t a i n e d i n the h i g h l y v i s c o u s phase IV l i q u i d c r y s t a l a t 20°C. The c o u p l i n g s a r e a l m o s t i d e n t i c a l t o t h o s e o b t a i n e d from m i n e r a l o i l . S i n c e n e i t h e r ENDOR nor NMR on p a r a m a g n e t i c m o l e c u l e s n o r m a l l y y i e l d d i r e c t i n f o r m a t i o n on t h e number o f n u c l e i p a r t i c i p a t i n g i n a g i v e n t r a n s i t i o n , i t was n e c e s s a r y t o use t h e o r e t i c a l arguments. For t e s t i n g t h e o r e t i c a l p r e d i c t i o n s , one needs a c c u r a t e measurements o f t h e magnitudes a s . w e l l as the knowledge o f the a b s o l u t e s i g n s o f t h e h y p e r f i n e c o u p l i n g s . ENDOR measurements y i e l d v e r y p r e c i s e v a l u e s whereas t h e e a r l i e r NMR s t u d i e s y i e l d a c c u r a t e s i g n i n f o r m a t i o n . The NMR s t u d i e s show t h a t t h e s i g n o f t h e l a r g e s t p r o t o n c o u p l i n g i s n e g a t i v e and t h a t o f the s m a l l e s t one i s p o s i t i v e . R e s u l t s o f the t h e o r e t i c a l c a l c u l a t i o n s ^ 5 7 ' 7 2 ' 7 5 p r e d i c t t h a t t h e p h e n y l - r i n g o r t h o p r o t o n s (atoms 1, 5, 6, 10, F i g 16) s h o u l d show the l a r g e s t h y p e r f i n e c o u p l i n g s w i t h n e g a t i v e s i g n s . They a l s o i n d i c a t e t h a t t h e s m a l l e s t , p o s i t i v e c o u p l i n g s h o u l d be a s s i g n e d t o t h e meta p r o t o n s (atoms 2, 4, 7, 9) and t h a t t h e para p r o t o n s ( 3 , 8) s h o u l d have c o u p l i n g s T a b l e 5. H y p e r f i n e c o u p l i n g s and s p i n d e n s i t i e s o b t a i n e d f o r DPPH by ENDOR and EPR Atom # Type S p l i t t i n g (Gauss) ( a i ) S p i n D e n s i t i e s E x p e r i m e n t a l ' T h e o r e t i c a l d H y d r a z y l N i t r o g e n s +9.739 ± .007 c +7.933 ± .014' a 0.2713 0.322 0.2266 0.3751 1,6 5,10 3,8 11,12 2,7 4,9 o r t h o phenyl o r t h o phenyl and par a phenyl meta p i c r y l meta phenyl -2.006 ± .002" -1.895 ± .002° +1 .117 ± .002b +1.016 ± .002* +0.754 ± .002^ +0.675 ± .002* 0.0749 0.0707 •0.0417 -0.0379 -0.0281 -0.0252 0.0822 0.0777 0.0714 0.0379 •0.0395 •0.0375 13,14 15 o r t h o n i t r o p a ra n i t r o 0.7 ± 0.3 C 1.0 ± 0.3 C 0.02 0.03 -0.0051 -0.0050 a J . S . Hyde e t a l . ELD0R [ r e f e r e n c e 7 0 ] . & T h i s work, ENDOR. c T h i s work, e s t i m a t e d from f i t t i n g . ^V.A. Gubanov e t a l . [ r e f e r e n c e 6 7 ] . e Q H = -26.8, Q N = 35.9, Q N = 24.6 G. KO 95 w i t h i n t e r m e d i a t e magnitudes and n e g a t i v e s i g n s . F o r the p i c r y l r i n g p r o t o n s (11, 12) t h e s i g n s o f t h e h y p e r f i n e c o u p l i n g s a r e p r e d i c t e d t o be p o s i t i v e w h i l e t h e i r magnitudes s h o u l d be l a r g e r t h a n t h o s e f o r t h e meta p r o t o n s on t h e phenyl r i n g s . Thus, i n T a b l e 5 t h e two l a r g e s t c o u p l i n g s can be a s s i g n e d t o t h e f o u r o r t h o p r o t o n s and t h e two para p r o t o n s on t h e phenyl r i n g s , t h e l a r g e r o f t h e s e two b e i n g a s s i g n e d to the o r t h o p r o t o n s (1 and 6) t o ag r e e w i t h t h e t h e o r e t i c a l p r e d i c t i o n o f Gubanov e t a l . ' The s m a l l e r o f t h e s e two c o u p l i n g s ( i e . , t h e c o u p l i n g o f -1.895 Gauss) i s a s s i g n e d t o the pa r a p r o t o n s ( 3 , 8) and th e two o r t h o p r o t o n s ( 5 , 10) on t h e phenyl r i n g s . The two s m a l l e s t c o u p l i n g s a r e l i k e w i s e a s s i g n e d t o t h e f o u r meta p r o t o n s on t h e phenyl r i n g s and the r e m a i n i n g c o u p l i n g s ( w i t h magnitudes ^ 1 G) a r e a s s i g n e d to t h e meta p r o t o n s (11, 12) on the p i c r y l r i n g s . F u r t h e r s u p p o r t f o r t h e s e a s s i g n m e n t s i s o b t a i n e d t h r o u g h o t h e r arguments based on m o l e c u l a r geometry and t h e i n t e n s i t i e s o f t h e o b s e r v e d ENDOR s i g n a l s . F o r a m o l e c u l e as l a r g e as DPPH, t h e ' i n n e r ' hydrogens ( e g . , atoms 4, 5, 9, and 10) a r e e x p e c t e d t o e x p e r i e n c e more s t e r i c h i n d r a n c e than t h e o u t e r members (atoms 1, 2, 6 and 7), r e s u l t i n g i n s l i g h t l y l a r g e r s p i n d e n s i t i e s on atoms " l , 2, 6, 7 as" compared t o t h o s e on "atoms 4, 5, 9 and 10" r e s p e c t i v e l y . The a s s i g n m e n t o f t h e same c o u p l i n g t o the o r t h o p r o t o n s ( 5 , 10) and t h e para p r o t o n s ( 3 , 8) i s s u p p o r t e d by t h e o b s e r v e d i n t e n s i t y r a t i o o f 1:2 f o r t h e two ENDOR l i n e s .showi t h e l a r g e s t c o u p l i n g . S i n c e t h e l a r g e s t f r e q u e n c y ENDOR s i g n a l has been a s s i g n e d t o t h e two o u t e r , o r t h o p r o t o n s (1,6) on the phenyl r i n g s , t h e o t h e r c l o s e l y s p a c e d ENDOR s i g n a l s ( h a v i n g t w i c e as much i n t e n s i t y ) must a r i s e from 4 p r o t o n s . Two o f t h e s e p r o t o n s must be the o t h e r o r t h o 96 p r o t o n s ( 5 , 10) and the o t h e r two s h o u l d be the para p r o t o n s ( 3 , 8) on the phenyl r i n g s . A d d i t i o n a l s u p p o r t f o r t h e assignment.was o b t a i n e d by s i m u l a t i n g t h e ENDOR spectrum u s i n g t h e A l . l e n d o e r f e r a n d M a k i T 2 c o r r e c t i o n f o r m u l a o f e q u a t i o n [ 3 0 ] . With a v a l u e o f T 2 ^ 2.2 x 1 0 ~ 7 s e c , o b t a i n e d from s e v e r a l ENDOR s i m u l a t i o n s , t h e o b s e r v e d i n t e n s i t i e s match w e l l w i t h t h e number o f p r o t o n s a s s i g n e d above. The i n t e n s i t y r a t i o o f 1:2 o f t h e two h i g h e s t f r e q u e n c y ENDOR l i n e s c o u l d a l s o be e x p l a i n e d by a s s i g n i n g them r e s p e c t i v e l y t o the two para p r o t o n s ( 3 , 8) and t h e f o u r o r t h o p r o t o n s ( 1 , 5, 6, 1 0 ) . T h i s would i m p l y t h a t t h e ' i n n e r ' and t h e ' o u t e r ' o r t h o p r o t o n s a r e a l l e q u i v a l e n t and t h a t f o r t h e phenyl r i n g s t h e s p i n d e n s i t i e s a t t h e pa r a p o s i t i o n s a r e l a r g e r than t h o s e a t t h e o r t h o p o s i t i o n s . T h e o r e t i c a l c a l c u l a t i o n s o f Gubanov e t a l , ' i n c l u d e d i n T a b l e 5, i n d i c a t e t h a t o r t h o phenyl s p i n d e n s i t i e s s h o u l d be l a r g e r than t h e para s p i n d e n s i t i e s , and t h a t a l l t h e f o u r o r t h o p r o t o n s s h o u l d n ot show t h e same h y p e r f i n e coup-l i n g . The c a l c u l a t i o n s a l s o i n d i c a t e t h a t t h e f o u r o r t h o p r o t o n s s h o u l d form i n t o p a i r s o f two e q u i v a l e n t p r o t o n s . Both t h e s e o b s e r v a t i o n s t h e r e f o r e s u p p o r t t h e a s s i g n m e n t g i v e n i n T a b l e 5. I t might be argued t h a t t h e r e s u l t s o f t h e o r e t i c a l c a l c u l a t i o n s may n o t be p r e c i s e enough t o b r i n g o u t t h e r a t h e r s m a l l d i f f e r e n c e between the s p i n d e n s i t i e s a t t h e o r t h o and t h e p a r a p o s i t i o n s and t h a t t h e o r t h o p r o t o n s m i ght be e q u i v a l e n t even w i t h i n t h e ENDOR r e s o l u t i o n o f 20 kHz. The ENDOR r e s u l t s do show t h a t t h e foim.meta p r o t o n s ( 2 , 7, 4, 8) on t h e phenyl r i n g s a r e c l e a r l y not e q u i v a l e n t , f o r m i n g a g a i n two p a i r s as p r e d i c t e d by t h e o r y . T h i s i n e q u i v a l e n c e i n the meta p r o t o n s i n d i c a t e s 97 t h a t t h e o r t h o p r o t o n s a l s o can be e x p e c t e d t o be i n e q u i v a i e n t . In the absen c e o f ENDOR r e s u l t s on s p e c i f i c a l l y d e u t e r a t e d samples, t h e a s s i g n -ment as shown i n T a b l e 5 i s thus t h e most r e a s o n a b l e . The l i n e s a s s i g n e d t o the meta p i c r y l p r o t o n s a r e v e r y s l i g h t l y s p l i t . T h i s i n e q u i v a l e n c e i m p l i e s t h a t t h e p i c r y l r i n g does n o t p o s s e s s an a x i s o f symmetry. The i n e q u i v a l e n c e c o u l d a r i s e i f t h e p i c r y l r i n g i s not s t a t i o n a r y b u t undergoes motion r e s u l t i n g i n t h e l o s s o f t h e t w o - f o l d a x i s f o r t h e m o l e c u l e . I f t h i s m otion i s slow enough t o be f r o z e n on t h e ENDOR (NMR) ti m e s c a l e M 0 ~ 7 s e c ) t h e two p r o t o n s would a p p e a r t o be non-e q u i v a l e n t . The s p l i t t i n g o f t h e s i g n a l a p p e a r s t o be tem p e r a t u r e - d e p e n d e n t but t h e s i g n a l - t o - n o i s e r a t i o d i d n o t p e r m i t a d e t a i l e d s t u d y o f the temperature-dependence o f t h i s l i n e . A t the low fm d e v i a t i o n (15 kHz) n e c e s s a r y t o o b s e r v e t h i s s p l i t t i n g , t h e a m p l i f i c a t i o n f a c t o r from t h e f i r s t L o c k - i n a m p l i f i e r i s not v e r y g r e a t . Above t h i s t e m p e r a t u r e t h e t o t a l ENDOR enhancement f a l l s and no s p l i t t i n g can be o b s e r v e d . NMR work on a , a ' - d i p h e n y l - e ( 2 , 4 , 6 - t r i n i t r o p h e n y l ) h y d r a z i n e by 7fi H e i d b e r g e t a l shows a c l e a r i n e q u i v a l e n c e o f t h e meta 3 phenyl p r o t o n s a t 0°C. T h i s s p l i t t i n g c o a l e s c e s a t 35°C t o a s i n g l e l i n e . In t h e h y d r a z i n e t h e p r o t o n has a l r e a d y removed t h e m o l e c u l a r a x i a l symmetry, but by a n a l o g y i n e q u i v a l e n c e i n t h e DPPH p i c r y l p r o t o n s i s not unex p e c t e d . The c o u p l i n g s o f T a b l e 5 were used t o compute t h e s p i n d e n s i t i e s a t t h e v a r i o u s atoms. The McConnell r e l a t i o n 7 7 a C H " W C H P C [55] where a i s t h e c o u p l i n g , p 7 7 i s i r - e l e c t r o n s p i n d e n s i t y a t t h e c a r b o n atom 98 and Q i s a c o n s t a n t w i t h a v a l u e o f 0 = 26.8 G f o r phenyl p r o t o n s . The v a l u e s Q N = 35.9 and = 24.6 were used f o r t h e s p i n d e n s i t i e s on t h e a 3 h y d r a z y l n i t r o g e n s . N i t r o groups c o u l d m o d i f y t h i s s i m p l e r e l a t i o n s h i p f o r t h e p i c r y l r i n g . A p p l i c a t i o n o f M c C o n n e l l ' s r e l a t i o n s h i p i n d i c a t e s c o n s i d e r a b l e s p i n d e n s i t y i s l e f t f o r t h e n i t r o g r o u p s . S p i n d e n s i t i e s from t h e H a r t r e e - F o c k c a l c u l a t i o n s o f Gubanov e t a l ' a r e i n c l u d e d i n T a b l e 5. These show t h a t t h e n i t r o - g r o u p s p i n d e n s i t y needs some improve-ment. The a n a l y s i s o f t h e EPR s p e c t r a a l s o shows t h a t t h e s e n i t r o - g r o u p d e n s i t i e s s h o u l d be somewhatthigher t h a n p r e d i c t e d by t h e H a r t r e e - F o c k c a l c u l a t i o n s . The EPR o f DPPH c l a r i f i e s some o f t h i s d i s c u s s i o n . No ENDOR l i n e s were o b t a i n e d f r o m t h e h y d r a z y l n i t r o g e n s ( t h e y a r e e x p e c t e d t o be o u t s i d e t h e c a p a c i t o r range) o r t h e p i c r y l n i t r o g r o u p s . Thus t o o b t a i n some v a l u e s f o r a^Q and t o check t h e a s s i g n m e n t shown i n T a b l e 5, a d e t a i l e d i n v e s t i g a t i o n o f the EPR o f DPPH s o l u t i o n s was made. F i g 16(a) shows t h e s i m p l e s t EPR sp e c t r u m o f s o l u t i o n s o f DPPH o b t a i n e d by u s i n g m i n e r a l o i l as s o l v e n t . The spectrum c o n s i s t s o f o n l y a f i v e - l i n e p a t t e r n and resembles t h a t o b t a i n e d from f r o z e n s o l u t i o n s o r t h a t f r o m u s i n g undegassed t h i n s o l v e n t s . The spectrum can be a n a l y z e d i n terms o f t h e h y p e r f i n e i n t e r a c t i o n o f t h e u n p a i r e d e l e c t r o n w i t h t h e two c e n t r a l n i t r o g e n s N and N . The c o u p l i n g c o n s t a n t s o b t a i n e d a r e CX p c o n s i s t e n t w i t h t h o s e o f T a b l e 5. The l i n e s h a p e s can be e x p l a i n e d i n terms o f t h e f r e e z i n g o f t h e motion on t h e EPR time s c a l e because o f t h e h i g h v i s c o s i t y o f t h e s o l v e n t u s ed. A complex h y p e r f i n e p a t t e r n was o b s e r v e d when c a r e f u l l y degassed 99 and low v i s c o s i t y s o l v e n t s were used. A spectrum t a k e n a t room temper-a t u r e , w i t h n-heptane as s o l v e n t i s shown i n F i g 1 7 ( a ) . The spectrum c o n s i s t s o f a l a r g e number o f n e a r l y e q u i d i s t a n t l i n e s w i t h a s p a c i n g o f about 0.35 6 superimposed on a b a s i c f i v e - l i n e p a t t e r n . The f i v e -l i n e s t r u c t u r e can be a s s i g n e d t o t h e h y p e r f i n e s t r u c t u r e from t h e two c e n t r a l n i t r o g e n s . The l a r g e number o f t h e e q u a l l y spaced l i n e s must a r i s e from t h e p r o t o n and t h e n i t r o x y l n i t r o g e n h y p e r f i n e i n t e r a c t i o n . S i n c e no r e c o g n i z a b l e i n t e n s i t y p a t t e r n was o b s e r v e d , i t was o b v i o u s t h a t computer s i m u l a t i o n o f t h e EPR spectrum would be n e c e s s a r y f o r a c c u r a t e a n a l y s i s . To o b t a i n a c c u r a t e s i m u l a t e d EPR s p e c t r a , i t was n e c e s s a r y t o guess t h e h y p e r f i n e c o u p l i n g s o f t h e n i t r o x y l n i t r o g e n s . These c o u p l i n g s were e s t i m a t e d by a n a l o g y w i t h n i t r o x y l n i t r o g e n coup-l i n g s f o r s i m i l a r systems and w i t h t h e h e l p o f t h e a v a i l a b l e t h e o r e t i c a l c a l c u l a t i o n s . Comparison o f the ENDOR r e s u l t s w i t h t h o s e o b t a i n e d t h r o u g h EPR 78 s t u d i e s on t h e p - n i t r o t r i p h e n y l methyl r a d i c a l shows t h a t t h e s p l i t t i n g f o r t h e n i t r o g e n a t t h e para p o s i t i o n (15) on t h e p i c r y l r i n g s i s ^  0.75 G, f a i r l y c l o s e t o t h a t f o r t h e meta p r o t o n s (2, 4) on the phenyl r i n g s . A n alogous i n f o r m a t i o n on a t r i n i t r o a r o m a t i c r a d i c a l i s a v a i l a b l e o n l y f o r 79 t h e s y m m e t r i c a l t r i n i t r o b e n z e n e r a d i c a l a n i o n . T r i n i t r o b e n z e n e cannot be compared d i r e c t l y w i t h t h e p i c r y l r i n g , but t h e EPR r e s u l t s do s u g g e s t t h a t t h e c o u p l i n g s f o r t h e o r t h o n i t r o g e n s a r e ^ 1 t o 2 G. T h i s c o n c l u -s i o n i s f u r t h e r s u p p o r t e d by computer s i m u l a t i o n s t u d i e s on t h e EPR spectrum. In DPPH, i f the a s s i g n m e n t i s c o r r e c t , and a l m o s t a l l t h e p r o t o n s a r e d i f f e r e n t , t h e r e s h o u l d be a l a r g e number o f l i n e s 100 (b) Simulation w w 10 Gauss F i g u r e 17. ' (a) EPR o f (10 _ l tM) DPPH i n o u t g a s s e d n-heptane a t 20°C and (b) S i m u l a t e d EPR o f DPPH u s i n g h y p e r f i n e c o u p l i n g s from ENDOR. 101 (3x3x3x3x3x3x2x2x5x3 = 131,220). The s i m u l a t i o n would be e x p e n s i v e u n l e s s s p e c i a l t e c h n i q u e s were used. I n i t i a l s i m u l a t i o n s made w i t h no n i t r o group s p l i t t i n g s do not resemble t h e e x p e r i m e n t a l s p e c t r a e x c e p t a t l a r g e l i n e w i d t h s . A l l o f t h e s i m u l a t i o n s done on a s i n g l e h y d r a z y l n i t r o g e n e n v e l o p e u s i n g t h e ENDOR c o n s t a n t s f i t w e l l w i t h a l m o s t any p arameter v a r i a t i o n . With such a l a r g e number o f l i n e s (26,244) i n each o f t h e s e e n v e l o p e s , i t was n e c e s s a r y t o s i m u l a t e t h e e n t i r e s p ectrum each t i m e , as l i n e s c o u l d o v e r l a p as f a r as 10 G away from an e n v e l o p e c e n t r e . S i m u l a t i o n time was saved by n o t i n g t h a t , w i t h i n a l i n e w i d t h , t h e r e a r e n o t 6 but 3 n o n - e q u i v a l e n t p r o t o n c o u p l i n g c o n s t a n t s (84 p r o t o n l i n e s i n s t e a d o f 729 per h y d r a z y l n i t r o g e n l i n e ) . S i n c e a ^ ( o r t h o ) £ a ^ ( p a r a ) ^ 2 a U i ( m e t a - p i c r y l ) , t h i s number can be r e d u c e d by c o n s t r u c t i n g an i n i t i a l s t i c k d i a g r a m w i t h s p l i t t i n g o f a ^ ( m e t a - p i c r y l ) o n l y , g i v i n g r i s e t o 45 l i n e s i n s t e a d o f 84. I n c l u s i o n o f a 0.7 G s p l i t t i n g f o r t h e p a r a n i t r o group by t h e same p r e c o n s t r u c t i o n t e c h n i q u e (aj^Q ( p a r a ) ^ a^(meta)) improved the f i t o f the s i m u l a t e d s p ectrum c o n s i d e r a b l y . V a r i o u s o t h e r v a l u e s o f n i t r o group c o u p l i n g s were t r i e d , based on c h e m i c a l and m o l e c u l a r - s t r u c t u r a l arguments. The b e s t improvement was o b t a i n e d by a s s i g n i n g 0.7 G f o r the 2 o r t h o n i t r o groups and 1.0 G f o r t h e para n i t r o g r o u p s . A t t h i s p o i n t , the d i f f e r e n c e s between t h e s p e c t r a were v e r y s m a l l . The EPR s p e c t r u m s i m u l a t e d u s i n g t h e s e c o n s t a n t s i s shown i n F i g 1 7 ( b ) . The agreement i s c o n s i d e r e d t o be good i n view o f the u n c e r t a i n t y i n t h e n i t r o group c o u p l i n g s and t h e s o l v e n t and tempera-102 t u r e e f f e c t on the l i n e shape. N e a r l y 70 s i m u l a t i o n s were r e q u i r e d t o o b t a i n t h i s f i t . 80 Very r e c e n t work by Gubanov e t a l . does n o t s u p p o r t t h i s a s s i g n m e n t , and i n d i c a t e s t h a t t h e n i t r o group v a l u e s s h o u l d be much l o w e r , about 0.38 G. These a u t h o r s have a p p l i e d t h e t e c h n i q u e o f F o u r i e r t r a n s -f o r m a t i o n t o the a n a l y s i s o f t h e EPR sp e c t r u m . T h i s t e c h n i q u e was o r i g -81 82 i n a l l y i n t r o d u c e d by Dobryakov and i n d e p e n d e n t l y by S i l s b e e . In t h i s t e c h n i q u e , i n s t e a d o f a n a l y s i s o f an o b s e r v e d spectrum i n the c o n v e n t i o n a l way, t h e spectrum i s c o n v e r t e d t o i t s F o u r i e r t r a n s f o r m which i s then compared w i t h t h e F o u r i e r t r a n s f o r m o f t h e e x p e c t e d s p e c t r u m . The t h e o r -e t i c a l s y n t h e s i s o f t h e F o u r i e r t r a n s f o r m o f an EPR spectrum w i t h a g i v e n s e t o f h y p e r f i n e c o u p l i n g s t u r n s out to be s i m p l e r than t h e s y n t h e s i s o f th e spectrum i t s e l f . From t h e t r a n s f o r m e d spectrum showing t h e b e s t f i t , t h e EPR spectrum i s r e g e n e r a t e d and compared w i t h t h e o b s e r v e d one. T h i s t e c h n i q u e i s e s p e c i a l l y s u i t a b l e f o r computing methods but r e q u i r e s a d i g i t i z e d EPR spectrum. The e x c e l l e n t agreement between t h e i r o b s e r v e d and t h e s i m u l a t e d EPR s p e c t r a o f DPPH de m o n s t r a t e s t h a t t h e t e c h n i q u e i s q u i t e p r o m i s i n g f o r a n a l y s i s o f c o m p l i c a t e d EPR s p e c t r a . A c o m p a r i s o n o f h y p e r f i n e c o u p l i n g s e s t i m a t e d f o r DPPH u s i n g t h i s t e c h n i q u e w i t h t h o s e measured d i r e c t l y t h r o u g h ENDOR and ELDOR b r i n g s o u t some o f t h e l i m i t a t i o n o f t h e F o u r i e r - t r a n s f o r m method. The c o u p l i n g s f o r t h e c e n t r a l n i t r o g e n s , N^ and N^, have been measured a c c u r a t e l y i n s o l u t i o n by Hyde e t a l 7 0 t h r o u g h ELDOR, benzene b e i n g used as s o l v e n t . 80 The v a l u e s e s t i m a t e d by Gubanov e t a l . f o r N and N„ a r e s m a l l e r than t h e ELDOR v a l u e s by a p p r o x i m a t e l y 0.4 G, which i s t h e s m a l l e s t s e p a r a -103 t i o n i n t h e o b s e r v e d EPR spectrum. The s o l v e n t used by Gubanov e t a l was a b e n z e n e - t e t r a h y d r o f u r a n m i x t u r e , which i s perhaps s i m i l a r t o benzene, used i n the ELDOR e x p e r i m e n t s . Of c o u r s e t h e p r e s e n c e o f d i s s o l v e d oxygen c o u l d a l t e r t h e c o u p l i n g s , but t h e samples were r e p o r t e d t o be t h o r o u g h l y d e g a s s e d . The absence o f oxygen i s a l s o i n d i c a t e d by the v e r y h i g h r e s o l u t i o n i n t h e o b s e r v e d EPR s p e c t r a . Small d i f f e r e n c e s a r e a l s o a p p a r e n t i n t h e p r o t o n c o u p l i n g s 80 measured by ENDOR and t h o s e r e p o r t e d by Gubanov e t a l . F o r the ENDOR work m i n e r a l o i l was us e d , not benzene. T h i s might a c c o u n t f o r some s m a l l e r d e v i a t i o n s but some c o u p l i n g s d i f f e r by as much as 40 t o 50%. The l a r g e s t d i f f e r e n c e , f o r meta p i c r y l p r o t o n s , i s a g a i n 0.4 G. I t i s c u r i o u s t h a t both f o r the c e n t r a l n i t r o g e n s and some p r o t o n s t h e d i f f e r e n c e t u r n s o u t t o be equal t o t h e s m a l l e s t s e p a r a t i o n o b s e r v e d i n t h e EPR s p e c t r u m . One r e a s o n f o r t h e s e d i s c r e p a n c i e s might be t h a t , as w i t h o t h e r c u r v e s - f i t t i n g p r o c e d u r e s , t h i s t e c h n i q u e m i ght admit a f a m i l y o f 81 s o l u t i o n s . T h i s was i n d i c a t e d by Dobryakov f o r a s i m p l e c a s e o f h y p e r f i n e s t r u c t u r e from one p r o t o n and one n i t r o g e n n u c l e u s . Gubanov e t a l s t a t e t h a t t h e y o b t a i n e d two s e t s o f h y p e r f i n e c o u p l i n g s , and both s e t s gave e q u a l l y good f i t s t o the o b s e r v e d EPR spectrum. One o f t h e s e t s was then e l i m i n a t e d w i t h t h e h e l p o f an NMR spectr u m . The d i f f i c u l t y i s i n c r e a s e d by t h e f a c t t h a t here t h e extreme l i n e s i n t h e wings o f t h e s p e c t r a , u s u a l l y v e r y d i f f i c u l t ' ' t o o b s e r v e , become q u i t e c r i t i c a l f o r co m p a r i s o n . Thus i t i s c o n c e i v a b l e t h a t a complex o b s e r v e d h y p e r f i n e p a t t e r n might be d e s c r i b e d by s e v e r a l s e t s o f h y p e r f i n e c o u p l i n g s , o n l y one o f which i s t h e t r u e one. 104 In o r d e r t o r e s o l v e some o f t h e s e d i f f i c u l t i e s , some s y n t h e t i c ENDOR s p e c t r a were computed, and t h e s e a r e shown i n F i g 18. F i g 18(a) i s s i m u l a t e d w i t h t h e c o n s t a n t s o f T a b l e 5 and a T 2 = 2 x 1 0 " 7 s e c , 18(b) i s t h e measured ENDOR, 18( c ) and 18(d) a r e w i t h t h e v a l u e s r e p o r t e d 80 by Gubanov e t a l . These a u t h o r s e l i m i n a t e d t h e v a l u e s o f 18(d) because t h e l i n e marked w i t h a s t a r d i d not appear i n NMR s t u d i e s . Comparison w i t h t h e ENDOR o f 18(c) shows t h e r e i s no c o u p l i n g w i t h t h i s v a l u e and i t can be e l i m i n a t e d by ENDOR as w e l l . The v a l u e s f o r F i g 18(c) can thus be e l i m i n a t e d as w e l l : t h e r e i s no c e n t r a l l i n e n e a r 12 and 15 MHz.and, more i m p o r t a n t l y , t h e i n t e n s i t y r a t i o o f t h e o u t e r • l i n e s i s . r e v e r s e d . T h e r e a r e a l s o s m a l l d i f f e r e n c e s i n t h e p o s i t i o n s o f a l l t h e l i n e s . I t was o r i g i n a l l y t h o u g h t t h a t t h i s r e v e r s a l o f i n t e n s i t i e s m i ght be a t y p o g r a p h i c a l e r r o r , but t h e EPR can o n l y be s i m u l a t e d w i t h t h i s i n t e n s i t y r a t i o : r e v e r s i n g t h e c o u p l i n g s f o r t h e o r t h o and para p r o t o n s g i v e s an EPR which b e a r s no re s e m b l a n c e t o the measured spectrum o f F i g 1 7 ( a ) , w h i l e Gubanov's c o n s t a n t s used f o r 1 8 ( c ) and (d) r e p r o d u c e e x a c t l y t h e EPR spectrum o f F i g 1 7 ( a ) . T h i s d i s c u s s i o n shows t h a t t h e h y p o t h e s i s o f Dobryakov i s c o r r e c t : f o r t h i s c a s e a t l e a s t t h e r e i s a f a m i l y o f s o l u t i o n s . The p a r t i c u l a r s o l u t i o n chosen a g r e e s p e r f e c t l y w i t h t h e EPR and (b r o a d l i n e ) NMR, b u t i t does not a g r e e w i t h t h e ELDOR o r ENDOR work on DPPH: t h e number o f l i n e s f o r ENDOR i s i n c o r r e c t , and t h e f r e q u e n c i e s f o r both ENDOR and ELDOR do n o t a g r e e w i t h t h i s s o l u t i o n . In t h e absence o f ENDOR on t h e n i t r o groups o r n i t r o g e n NMR, t h e c o n s t a n t s o f T a b l e 5 a r e f e l t t o be t h e b e s t v a l u e s f o r t h e n i t r o g r o u p s : a ^ = 0.7 and 1.0 G.' C o n s i d e r i n g t h e l i m i t a t i o n s o f t h e v a r i o u s t e c h n i q u e s , i t a p p e a r s t h a t t h e ENDOR t e c h -n i q u e combined w i t h t h e F o u r i e r t r a n s f o r m method c o u l d be a v e r y powerful DPPH-ENDOR a) b) c-Gi) d-G2) J 105 F i g u r e 18. S i m u l a t e d ENDOR s p e c t r a o f DPPH u s i n g : (a) DPPH c o u p l i n g s o b t a i n e d i n t h i s work (b) Measured ENDOR s p e c t r u m o f DPPH ( c ) 1 s t s e t o f DPPH c o u p l i n g s f r o m r e f 80-(d) 2nd s e t o f DPPH c o u p l i n g s f r o m r e f 80. 106 t o o l f o r u n r a v e l l i n g v e r y complex h y p e r f i n e s t r u c t u r e i n EPR. The l i n e i n t e n s i t y f o r t h e DPPH ENDOR l i n e s a t 12 and 15 MHz i s much l a r g e r t h a n e x p e c t e d , even w i t h t h e T 2 c o r r e c t i o n f o r m u l a , f o r j u s t t h e two p r o t o n s o f t h e p i c r y l r i n g . The p o s s i b l e e x p l a n a t i o n s f o r t h i s e f f e c t i n c l u d e r f c o h e r e n c e e f f e c t s ' ? the' p r e s e n c e o f pa r a m a g n e t i c im-p u r i t i e s i n t h e sample, o r p o s s i b l y i o n p a i r s , and unusual enhancement mechanisms. T h i s l i n e always o c c u r r e d i n t h e DPPH ENDOR, even when t h e f i n e r e s o l u t i o n o f F i g 16 was n o t o b t a i n e d , and t h e i n t e n s i t y r a t i o o f t h e t h r e e groups was always about t he same a t v a r i o u s t e m p e r a t u r e s , i n m i n e r a l o i l and i n l i q u i d c r y s t a l . I f t h e peaks a t 12 and 15 MHz a r e a p a r a m a g n e t i c i m p u r i t y ; then t h e above o b s e r v a t i o n s show t h a t i t must have a s i m i l a r EPR g v a l u e t o DPPH, and must always o c c u r i n about t h e same c o n c e n t r a t i o n as t h e DPPH i n a l l t he v a r i o u s s o l u t i o n s s t u d i e d . T h i s s o r t o f ' i m p u r i t y ' i s un-l i k e l y , and i f t h e l i n e s r e a l l y do n o t b e l o n g t o DPPH, t h e n an ' i o n - p a i r ' h y p o t h e s i s i s more l i k e l y . An i o n p a i r i n v o l v i n g DPPH would always produce a second s e t o f l i n e s i n a b o u t t h e same r a t i o t o the DPPH l i n e s i n a l l s o l u t i o n s a t v a r i o u s t e m p e r a t u r e s . I f t h i s i s t r u e , i t i s re m a r k a b l e t h a t a l l t he c o n s t a n t s measured do r e p r o d u c e so w e l l t he EPR measured i n o u t g a s s e d t h i n s o l v e n t s , such as n-heptane, where no p a i r i n g has e v e r been p o s t u l a t e d . The i n t e n s i t y o f l i n e s a r i s i n g f r o m p a i r f o r m a t i o n c o u l d be e x p e c t e d t o change w i t h t e m p e r a t u r e , and t h i s was n e v e r o b s e r v e d , a l t h o u g h t h e range o f t e m p e r a t u r e s o v e r which m i n e r a l o i l g i v e s ENDOR enhancement i s about 60°C, In both o f t h e s e c a s e s , i t i s noted t h a t t h e r e s t o f t h e spectrum, 107 o m i t t i n g t h i s l i n e , c o n s i s t s o f l i n e s w i t h a c l e a r i n t e n s i t y r a t i o o f 2:2:4:2 and thus both o f t h e above h y p o t h e s e s demand t h a t 2 p r o t o n s a r e m i s s i n g f r o m t h e ENDOR spectrum. S i n c e t h e r e s t o f t h e spectrum can be a d e q u a t e l y a s s i g n e d from t h e o r e t i c a l c a l c u l a t i o n s a l o n e , i t i s most prob-a b l e t h a t t h e two c a s e s above a r e i n v a l i d , and t h a t t h e l i n e s a t 12 and 15 MHz do b e l o n g t o some p a r t o f the DPPH m o l e c u l e . The p o s s i b i l i t y o f c o h e r e n c e e f f e c t s on t h i s l i n e a l o n e a r e remote. T h i s s t u d y was done w i t h a h i g h power r f s o u r c e , but i t was u s u a l l y o p e r a t i n g a t 250 W and t h e u s u a l i n t e r n a l r f f i e l d was around 15 G. Co-herence e f f e c t s do show up as l i n e s p l i t t i n g i n t h e ENDOR spectrum, but a r e not n o r m a l l y o b s e r v e d u n t i l f i e l d s o f 30 G o r more a r e used. Coherence has been o b s e r v e d on t h i s s p e c t r o m e t e r , b u t o n l y w i t h a low-t e m p e r a t u r e s o l i d sample ( s u c r o s e ) , and i t i s d o u b t f u l i f t h e r f f i e l d can be d r i v e n h i g h enough t o see c o h e r e n c e i n l i q u i d s w i t h t h e . p r e s e n t a m p l i f i e r . Coherence e f f e c t s a r e u s u a l l y seen when a l i n e i s overpowered by e x c e s s i v e r f a t l e a s t 2 t o 4 dB above t h e a p p e a r a n c e p o i n t o f the l i n e . When t h e r f f i e l d used f o r DPPH was lowered t h e e n t i r e spectrum v a n i s h e d i n t o t h e background n o i s e . With t h e above c o n s i d e r a t i o n s , i t i s u n l i k e l y t h a t t h e unusual i n t e n s i t y and s p l i t t i n g o f t h i s l i n e i s due t o any r f c o h e r e n c e e f f e c t s . The f i n a l p o s s i b i l i t y o f an unusual enhancement f a c t o r i s l e f t by e l i m i n a t i o n . T h i s seems t h e most l i k e l y : t h e s e l i n e s b e l o n g t o the p i c r y l . group hydrogens, on a n o t h e r p a r t o f t h e m o l e c u l e . A d i f f e r e n c e i n enhancement mechanism i s q u i t e p o s s i b l e , e s p e c i a l l y s i n c e t h e p r e s e n c e o f t h e N0 2 groups on t h e p i c r y l r i n g might a l t e r t h e r e l a x a t i o n pathways. M o t i o n a l e f f e c t s o f the r i n g might a l s o a c c o u n t f o r some o f t h e d i f f e r e n c e . 108 I t i s c o n c l u d e d t h a t the b e s t h y p o t h e s i s i s a s s i g n m e n t o f t h i s l i n e t o the p i c r y l h y d r o g e n s , w i t h a h i g h l y anomalous i n t e n s i t y . The complete enhancement pathway f o r t h i s l i n e i s not u n d e r s t o o d . I t i s n o t o b v i o u s from T a b l e 5, but t h e r e a r e many a c c i d e n t a l o v e r l a p s i n t h i s s p e c t r u m , and t h e s e can be seen i n T a b l e 6. The e q u a l i t i e s o f v a r i o u s c o u p l i n g s a r e w i t h i n a l i n e w i d t h , and A i s t h e o b s e r v e d s p a c i n g between s u c c e s s i v e l i n e s . The f i r s t two e q u a t i o n s show a c c i d e n t a l o v e r l a p s o f c o u p l i n g s , w h i l e t h e l a s t f o u r a r e o v e r l a p s o f d i s t a n c e s between t h e h y d r a z y l n i t r o g e n e n v e l o p e c e n t r e s , as shown i n t h e F i g u r e . T h i s T a b l e a l s o shows t h a t t h e r e i s an e x p e r i m e n t a l b a s i s f o r the ELDOR model o f Hyde e t a l . 7 0 To e x p l a i n t h e p r e s e n c e o f e x t r a ' l i n e s , c a l l e d ' c o m b i n a t i o n l i n e s ' t h e y assumed t h a t t h e d i f f e r e n c e i n t h e c o u p l i n g c o n s t a n t s between t h e two c e n t r a l n i t r o g e n s was equal t o some p r o t o n c o u p l i n g a ^ , i e . , t h a t a^ - a N = aH.. The d a t a o f T a b l e 6 shows t h a t t h i s r e q u i r e m e n t i s f u l f i l l e d by some o f t h e p r o t o n s on t h e o r t h o and para p o s i t i o n s o f t h e phenyl r i n g s , and l e n d s s u p p o r t t o t h i s model. I t i s now c l e a r why o n l y 130 l i n e s a r e o b s e r v e d , a l t h o u g h t h e r e a r e 131,220 l i n e s e x p e c t e d . I t i s because magnitudes o f most o f t h e c o u p l i n g s a r e e i t h e r v e r y n e a r l y equal o r m u l t i p l e s o f each o t h e r . I t i s emphasized t h a t t h e r e s o l u t i o n o f t h e EPR spectrum o f F i g 17(a) i s not l i m i t e d by e i t h e r t h e magnetic f i e l d m o d u l a t i o n f r e q u e n c y o r by c o n c e n t r a -t i o n o r d e o x y g e n a t i o n . I t i s here c o n c l u d e d t h a t any s m a l l e r , u n r e s o l v e d s t r u c t u r e i s s u b s t a n t i a l l y s m a l l e r than 0.08 G, t h e l i m i t s e t by t h e 100 kHz m o d u l a t i o n f r e q u e n c y used i n r e c o r d i n g t h i s s p e c t r u m . 109 TABLE 6 A c c i d e n t a l o v e r l a p s i n DPPH c o u p l i n g c o n s t a n t s 3 a H ( m e t a ) = a H ( p a r a ) = 2 a N ( ^ ( o r t h o ) = 3 a N Q 2 ( p a r a ) a H ( p a r a ) = a H ( o r t h o ) 2 a N - a^ = 3 a H ( p a r a ) = 9 a H ( m e t a ) a 3 a N - a N = 2 a H ( m e t a ) + A 3 a a N = 4 a H ( p a r a ) a = 5 a ^ ( p a r a ) - A = 14a^(meta) + A Where A i s t h e o b s e r v e d s p a c i n g 0.35 G) between s u c c e s s i v e l i n e s H y d r a z y l n i t r o g e n e n v e l o p e c e n t e r s no T h i s c o m p l e t e a n a l y s i s shows t h a t t h e r e i s some s p i n d e n s i t y everywhere i n t h e DPPH r a d i c a l . Comparison o f t h e d a t a o b t a i n e d ( T a b l e /? 7 71 7 0 7 C 5) w i t h t h e a v a i l a b l e t h e o r e t i c a l work ' ' ' shows t h a t more a c c u r a t e c a l c u l a t i o n s a r e n e c e s s a r y . In such a l a r g e m o l e c u l e , f r a g m e n t c a l c u l a -t i o n would p r o b a b l y be t h e b e s t way t o r e p r o d u c e t h e a c c u r a t e ENDOR v a l u e s . T h e - : i n e q u i v a l e n c e o f the o r t h o and meta p r o t o n s i n d i c a t e s t h a t t h e e n t i r e m o l e c u l e i s t w i s t e d o r d i s t o r t e d . The i n e q u i v a l e n c e o f t h e two m e t a l p i c r y l p r o t o n s d e m o n s t r a t e s t h a t t h e r e i s dyn a m i c a l motion t h a t i s slow on t h e n u c l e a r time s c a l e o r t h a t t he p i c r y l r i n g i s a l s o t w i s t e d . I t i s a l s o p o s s i b l e t h a t t h e r e a r e two d i f f e r e n t s t a b l e p i c r y l r i n g c o n f i g u r a t i o n s . A l l o f t h e s e f a c t s must be c o n s i d e r e d i n any f u r t h e r t h e o r e t i c a l work on DPPH. The ENDOR r e s u l t s c o m p l e t e l y e x p l a i n t h e complex EPR h y p e r f i n e p a t t e r n t h a t i s o b s e r v e d i n h i g h l y o u t g a s s e d DPPH s o l u t i o n s . The key p o i n t i s t h a t t h e p r o t o n and n i t r o g e n c o u p l i n g s a r e e i t h e r equal o r m u l t i p l e s o f each o t h e r and o f the c o n s t a n t d i f f e r e n c e s between t h e s u c c e s s i v e EPR l i n e s . 4.3 P i c r y l - N - A m i n o C a r b a z y l (PAC) The s t a b l e f r e e r a d i c a l , p i c r y l - N - a m i n o c a r b a z y l , whose s t r u c t u r e and numbering scheme a r e shown i n F i g 19, p o s s e s s e s one o f th e most a n i s o t r o p i c e l e c t r o n i c g - t e n s o r s known i n o r g a n i c f r e e r a d i c a l s . I t has been h y p o t h e s i z e d by Hyde t h a t a l a r g e a n i s o -t r o p y i n t h e g - f a c t o r might have been p a r t o f the d i f f i c u l t y i n o b t a i n -i n g ENDOR enhancements i n p r e v i o u s s t u d i e s on DPPH. In o r d e r t o see i f t h e t e c h n i q u e s t h a t produced a m e a s u r a b l e enhancement i n DPPH were a p p l i c a b l e , an ENDOR s t u d y o f PAC was done. The s i m i l a r i t y between DPPH and PAC was u s e f u l f o r a s s i g n -ment o f o b s e r v e d c o u p l i n g s t o s p e c i f i c p r o t o n s i n both m o l e c u l e s . P r e v i o u s work on PAC i s n o t n e a r l y as e x t e n s i v e as on DPPH. The a l m o s t i d e n t i c a l s t r u c t u r e g i v e s r i s e t o a s i m i l a r EPR, w h i c h , l i k e DPPH, has n o t been c o m p l e t e l y i n t e r p r e t e d . The ENDOR t e c h n i q u e as shown f o r DPPH g i v e s a c c u r a t e c o u p l i n g s , adequate a s s i g n m e n t s ( i f t h e T 2 c o r r e c -t i o n i s i n c l u d e d ) and some i n f o r m a t i o n on t h e m o l e c u l a r d i s t o r t i o n , PAC, as shown i n F i g 19, has an e x t r a bond h o l d i n g t h e phenyl r i n g s t o g e t h e r and c o u l d be e x p e c t e d t o be more r i g i d and symmetric. E a r l i e r c.q RA pc, EPR s t u d i e s on PAG, ' ' m o s t l y i n c o n j u n c t i o n w i t h DPPH showed no 59 p r o t o n h y p e r f i n e s t r u c t u r e . L o r d and B l i n d e r d i d e s t a b l i s h t h a t t h e broad l i n e s p e c t r a o b s e r v e d c o u l d a r i s e o n l y i f t h e s p i n d e n s i t y on one c e n t r a l n i t r o g e n was do u b l e t h a t on the o t h e r . T h i s i s i n agreement w i t h l a t e r work, and can be c o n t r a s t e d w i t h r e s u l t s on DPPH, where t h e two c e n t r a l n i t r o g e n s a r e a l m o s t e q u i v a l e n t . F i g u r e 19. P i c r y l - n - a m i n o c a r b a z y l r a d i c a l , showing numbering s y s t e m and a p p r o x i m a t e c o u p l i n g s o b t a i n e d ( 6 ) . 113 R e c e n t l y , Makino e t a l o b t a i n e d a p a r t i a l l y r e s o l v e d spectrum o f PAC i n o u t g a s s e d c h l o r o f o r m . The r e s o l u t i o n was i n s u f f i c i e n t f o r a d e t a i l e d a n a l y s i s , b u t v a l u e s o f 11.1 and 6.0 Gauss were o b t a i n e d f o r t h e c e n t r a l n i t r o g e n s . The r e s t o f the c o n s t a n t s p r e d i c t e d by Makino e t a l do n o t s i m u l a t e t h e o b s e r v e d spectrum a t a l l e x c e p t a t l a r g e l i n e w i d t h s . For samples which gave optimum ENDOR enhancements, t h e room t e m p e r a t u r e EPR spectrum ( i n m i n e r a l o i l ) c o n s i s t s o f f o u r main peaks w i t h t h r e e s m a l l e r ones i n between as shown i n F i g 20. A sample w i t h c o n c e n t r a t i o n s t r o n g enough t o g i v e a s i g n a l - t o - n o i s e g r e a t e r than 200:1 s t i l l showed microwave s a t u r a t i o n a t around 50 mW i n c i d e n t power. T h i s s p e c t r u m a r i s e s from t h e h y p e r f i n e i n t e r a c t i o n o f t h e u n p a i r e d e l e c t r o n w i t h t h e two n i t r o g e n s N^ and N^ ", w i t h c o u p l i n g s i n the r a t i o o f a p p r o x i m a t e l y 2:1. The o b s e r v e d l i n e s h a p e s r e s u l t from t h e s l o w i n g down o f m o l e c u l a r motion because o f the h i g h v i s c o s i t y o f 59 m i n e r a l o i l , as has a l s o been r e p o r t e d f o r o t h e r v i s c o u s ; s o l v e n t s . F i g u r e 21(a) and (b) show ENDOR s p e c t r a o f t h i s sample o f PAC i n m i n e r a l o i l a t 340 K and 300 K. The enhancement i n c r e a s e s g r e a t l y a t t h e lower t e m p e r a t u r e as p r e d i c t e d by e q u a t i o n [25], but t h e spectrum becomes f a r more c o m p l i c a t e d . Below room t e m p e r a t u r e the enhancement f a l l s as t h e . m i n e r a l o i l t u r n s g l a s s y , and a s i g n a l - t o -n o i s e o f o n l y 3:1 was o b t a i n e d . The spectrum c o n s i s t s o f t h r e e groups o f l i n e s around t h e f r e e p r o t o n v a l u e . U s i n g e q u a t i o n F7] , c o u p l i n g s were measured f o r both t e m p e r a t u r e s and a r e shown i n T a b l e 7. F i g u r e 20. EPR o f (10" 3M) PAC i n m i n e r a l o i l as used f o r ENDOR. F i g u r e 21. (a) ENDOR s p e c t r u m o f PAC i n m i n e r a l o i l a t 340K and (b) ENDOR s p e c t r u m o f same sample o f PAC a t ^  290K. The e n l a r g e m e n t shows t h e c e n t r a l l i n e s . TABLE 7 H y p e r f i n e coup!.ings-and s p i n d e n s i t i e s o b t a i n e d f o r PAC. s o l u t i o n s f r o m ENDOR and EPR Atom # Type C o u p l i n g s (Gauss) S p i n D e n s i t i e s 340 K 290 K e a H y d r a z y l N i t r o g e n s 11.1 6.0 + CM CM 11.1 6.0 + + , | , a , 0.309 0.244 1 5 P r o t o n s : a l p h a c a r b a z y l - l ; 9 2 4 + .005 -1.986 -1.908 + + .002 .002 0.0741 0.0712 3 7 gamma c a r b a z y l -1.809 + .005 -1.836 -1.762 + + .002 .002 0.0685 0.0657 9,10 meta p i c r y l +1.173 + .005 +1.170 + .002 -0.0436 2,4,6,8 b e t a and d e l t a c a r b a z y l +0.527 +0.406 + + .005 .005 +0.548 +0.462 +0.375 + + ' + .002 .002 .002 -0.0205 -0.0172 -0.0140 11,12 o r t h o n i t r o -- 0.8 + °- 2|(c) 0.2) . 0.014 13 para n i t r o -- 1.2 + 0.03 T 2 v ; v a l u e s ^0.1 y s e c ^0.05 y s e c (a) From EPR f i t t i n g s , i n agreement w i t h t h e v a l u e s r e p o r t e d by Makino e t a l . ( r e f e r e n c e 8 3 ) (b) C a l c u l a t e d u s i n g t h e McConnell e q u a t i o n w i t h t h e f o l l o w i n g p a r a m e t e r s , QH = - 2 6 . 8 , QNA = 3 5 . 9 , QNR = 2 4 . 6 G .67 s i g n s taken from t h e o r e t i c a l c a l c u l a t i o n s ^ , 7 2 and NMR r e s u l t s on DPPH.72, These v a l u e s a r e f o r t h e low temperature c o n s t a n t s . ( c ) From f i t t i n g EPR s i m u l a t i o n . (d) From ENDOR s i m u l a t i o n s , u s i n g e q u a t i o n 30,. 117 U s i n g t h e h i g h e r t e m p e r a t u r e c o u p l i n g s , s e v e r a l ENDOR s p e c t r a were s y n t h e s i z e d u s i n g QUICK as d e s c r i b e d i n s e c t i o n 2.7. Comparison o f e x p e r i m e n t a l and s i m u l a t e d s p e c t r a showed t h a t t h e s e c o u p l i n g s match t he l i n e p o s i t i o n s q u i t e w e l H and t h e i n t e n s i t i e s f i t w e l l i f a T 2 v a l u e o f . about 0.1 m i c r o s e c o n d i s used f o r e q u a t i o n [ 3 0 ] . As the t e m p e r a t u r e i s lowered t o 300 K, t h e enhancement i n c r e a s e s , and two o f the t h r e e groups show a d d i t i o n a l s t r u c t u r e , F i g 2 1 ( b ) . T h i s i m p l i e s t h a t s e v e r a l o f t h o s e p r o t o n s which a r e magnet-i c a l l y e q u i v a l e n t a t h i g h e r t e m p e r a t u r e , become i n e q u i v a l e n t a t t h e lower t e m p e r a t u r e s . The c o u p l i n g s measured a t ^  300 K a r e a l s o i n c l u d e d i n t h e t a b l e . A comparison o f the s p e c t r a s i m u l a t e d from t h e s e c o u p l i n g s w i t h t h o s e o b s e r v e d e x p e r i m e n t a l l y showed t h a t here a l s o t h e o v e r l a p c o r r e c t i o n was n e c e s s a r y . The f i t o f the low t e m p e r a t u r e ENDOR s p e c t r a c o u l d n o t be made as good as t h a t o f t h e h i g h e r t e m p e r a t u r e o n es, b ut a s a t i s f a c t o r y f i t was o b t a i n e d by em p l o y i n g T 2 = 0.2 m i c r o s e c o n d s . As w i t h DPPH, t h e ENDOR measurements o f PAC were not s u f f i c i e n t f o r an unambiguous as s i g n m e n t o f the measured c o u p l i n g s t o v a r i o u s hydrogens i n the m o l e c u l e . T h e o r e t i c a l e s t i m a t e s o f t h e s e h y p e r f i n e c o u p l i n g s o r e x p e r i m e n t a l measurements t h r o u g h o t h e r t e c h n i q u e s ( e g. NMR) do n o t seem t o have been r e p o r t e d f o r PAC. The c o r r e s p o n d i n g f-J -J>p y o D C i n f o r m a t i o n was a v a i l a b l e on DPPH, * ' ' which i s f a i r l y s i m i l a r . The comparison o f the PAC d a t a w i t h t h a t o f DPPH can s e r v e as a check on t he as s i g n m e n t o f h y p e r f i n e c o u p l i n g s i n DPPH i t s e l f . The main d i f f e r e n c e i n the c h e m i c a l s t r u c t u r e s o f DPPH and PAC i s the p r e s e n c e o f an e x t r a C-C bond between the two phenyl r i n g s and t h e co n s e q u e n t l a c k o f two c o r r e s p o n d i n g hydrogens i n PAC. I f t h i s 118 e x t r a bond d i d not change t he geometry o f t h e p a r e n t m o l e c u l e a p p r e c i a b l y then t he ENDOR t r a n s i t i o n s f o r PAC would o c c u r a t t h e same f r e q u e n c i e s as t h o s e f o r DPPH, e x c e p t t h a t t h e t r a n s i t i o n s i n v o l v i n g t h e two ' e x t r a ' hydrogens i n DPPH would be a b s e n t i n t h e s p e c t r a f o r PAC. The e x t r a bond i n PAC would be e x p e c t e d t o cause f u r t h e r d i s t o r -t i o n o f the s t r u c t u r e o f t h e p a r e n t m o l e c u l e . T h i s was s t r o n g l y s u g g e s t e d by a m o l e c u l a r model o f PAC, c o n s t r u c t e d by u s i n g t he bond 87 l e n g t h s and bond a n g l e s r e p o r t e d f o r DPPH. I t i s t h e r e f o r e e x p e c t e d t h a t some o f the p r o t o n s which a r e m a g n e t i c a l l y e q u i v a l e n t i n DPPH would become i n e q u i v a i e n t i n PAC and as many as twenty ENDOR l i n e s m i g ht be e x p e c t e d f o r PAC. F i g 21 (b) shows t h a t s i x t e e n l i n e s can be r e s o l v e d . T h e r e i s an e x t r e m e l y narrow q u a r t e t o f l i n e s o f equ a l i n t e n s i t y a t 11 and 16 MHz where i n DPPH t h e r e were d o u b l e t s w i t h i n t e n s i t y 2:4. The 6 o r t h o and para, phenyl p r o t o n s i n DPPH were d i v i d e d i n t o 2 groups w i t h i n t e n s i t y r a t i o s o f 2:4; l o s s o f t h e 2 p r o -t o n s i n PAC would g i v e r i s e t o a d o u b l e t w i t h r a t i o 2:2 f o r t h e a l p h a and gamma p r o t o n s (see T a b l e 7 ) . The q u a r t e t o b s e r v e d i n PAC shows t h a t a t t h i s lower t e m p e r a t u r e , t he m o l e c u l e i s d i s t o r t e d and f r o z e n i n a c o n f i g u r a t i o n where the a x i a l symmetry i s l o s t . T h i s i s f u r t h e r s u p p o r t e d by t h e o b s e r v a t i o n i n the c a s e o f PAC o f a t r i p l e t (1:2:1) around 13 MHz where f o r DPPH o n l y a d o u b l e t (1:1) was o b s e r v e d . The o r i g i n o f t h i s t r i p l e t i n t h e b e t a and d e l t a c a r b a z y l l i n e s i s not c o m p l e t e l y u n d e r s t o o d and o n l y s p e c i f i c d e u t e r a t i o n can r e s o l v e which l i n e s a r i s e f r o m b e t a and which from d e l t a p r o t o n s . The most l i k e l y a s s i g n m e n t i s t h a t t h e two d e l t a p r o t o n s , b e i n g f a r t h e s t from t h e d i s t o r t i n g i n f l u e n c e o f t h e p i c r y l r i n g , a r e e q u i v a l e n t and 119 t h e b e t a p r o t o n s a r e q u i t e d i f f e r e n t , w i t h c o u p l i n g s o f .55 and .38 Gauss. I t i s a l s o p o s s i b l e , as w i t h DPPH, t h a t t h e r e i s a c c i d e n t a l o v e r l a p and t h a t t h e t r i p l e t i s an o v e r l a p o f two 1:1 d o u b l e t s . In e i t h e r c a s e , t h e m o l e c u l e must be h i g h l y d i s t o r t e d a t t h i s t e m p e r a t u r e . 83 Makino e t a l r e p o r t t h a t t h e v e r y b e s t r e s o l u t i o n i n t h e EPR o c c u r r e d i n o u t g a s s e d c a r b o n t e t r a c h l o r i d e . To check t h e above a s s i g n m e n t , a h i g h r e s o l u t i o n EPR i n v e r y c a r e f u l l y o u t g a s s e d CC1^ was r e c o r d e d , and i s shown i n F i g 2 2 ( a ) . T h i s resembles t h e spectrum 83 r e p o r t e d by Makino e t a l . S i n c e the spectrum i s too complex f o r d i r e c t a n a l y s i s , a s i m u l a t i o n was attempted w i t h t h e ENDOR c o n s t a n t s , I t may be noted t h a t s i n c e t h e EPR was run i n C C l ^ b u t t h e ENDOR i n m i n e r a l o i l , i t i s not t o be e x p e c t e d t h a t t h e ENDOR c o n s t a n t s would g i v e an e x a c t f i t t o the o b s e r v e d EPR spectrum. I n i t i a l s i m u l a t i o n showed t h a t i t was n e c e s s a r y t o i n c l u d e some h y p e r f i n e s p l i t t i n g from t h e n i t r o g r o u p s , as t h e s p e c t r a were a g a i n n o t even c l o s e l y r e p r o d u c e d w i t h o u t t h e s e c o n s t a n t s . The f i n a l s i m u l a t i o n , u s i n g t h e " h i g h t e m p e r a t u r e " ENDOR c o n s t a n t s and n i t r o v a l u e s o f 0.8 and 1.2 G, i s shown i n F i g 2 2 ( b ) . The f i t i s s a t i s f a c t o r y c o n s i d e r i n g t h a t t h i s s p ectrum was s i m u l a t e d w i t h some 77,000 l i n e s . The anomalous s a t u r a t i o n b e h a v i o u r o f t h e wing l i n e s even a t 1 mW p r e v e n t s a p e r f e c t s i m u l a t i o n o f t h e wings . The v a l u e s o f 0.8 and 1.2 Gauss needed f o r s i m u l a t i o n w i t h t h e n i t r o groups a g a i n do n o t resemble t h o s e p r e d i c t e d by Gubanov e t on a l f o r DPPH but do g i v e t h e b e s t f i t f o r t h e o b s e r v e d EPR spectr u m . T h i s argument has been f u l l y d i s c u s s e d i n s e c t i o n 4.2. In the absence F i g u r e 22. (a) EPR o f (lO ' V l ) PAC i n o u t g a s s e d C C I 4 a t 20°C and (b) S i m u l a t e d EPR o f PAC u s i n g t h e h i g h e r t e m p e r a t u r e h y p e r f i n e c o u p l i n g s o b t a i n e d by ENDOR. 121 o f s p e c i f i c NMR o r ENDOR on t h e n i t r o g r o u p s , t h e s e h i g h v a l u e s a r e th e b e s t t h a t a r e c o n s i s t e n t w i t h t h e ENDOR on t h e p r o t o n s . As w i t h DPPH, t h e v a l u e s o f T a b l e 7 have s e v e r a l a c c i d e n t a l o v e r l a p s between t h e c o u p l i n g s and t h e c e n t r e s o f t h e c a r b a z y l e n v e l o p e s , A few o f t h e s e o v e r l a p s a r e shown i n T a b l e .8. TABLE 8 Some a c c i d e n t a l o v e r l a p s i n PAC c o n s t a n t s 21 a 2 j i+ = a^ a 20 a 2 > 4 = a N - a N = 5 a 1 > 5 + A a 3 2 a 6 j 8 = 2 a N - a N = a Q _ N ( ) 3 a * 3 a 9 5 l 0 = 2 a 3 } 7 = 3 a p _ N 0 2  1 9 a 9 , i o = 2 a^  a 1 1 a i , 1 5 = 2 a^  where A i s t h e o b s e r v e d l i n e s p l i t t i n g . T h i s i m p l i e s t h a t an ELDOR spectrum o f PAC would a l s o show the " c o m b i n a t i o n " l i n e s , and thus s h o u l d s e r v e as a f u r t h e r check on Hyde's m o d e l 7 0 f o r t h e ELDOR enhancements. These o v e r l a p s a l s o e x p l a i n why o n l y about 100 l i n e s a r e o b s e r v e d even though about 77,000 l i n e s may be e x p e c t e d i n t h e t o t a l EPR spectrum o f t h i s r a d i c a l . Under t h e 122 same c o n d i t i o n s , s i g n a l s f r o m PAC were found a t t e m p e r a t u r e s h i g h e r than t h o s e a t which DPPH and BDPA y i e l d e d optimum s i g n a l s . T h i s might be due t o t h e r a t h e r l a r g e a n i s o t r o p y i n t h e e l e c t r o n i c g - f a c t o r f o r PAC, s i n c e t h i s i s perhaps t h e main d i f f e r e n c e i n t h e mag n e t i c p r o p e r -t i e s o f t h e s e r a d i c a l s . PAC has been found t o be d i s t o r t e d such t h a t the m o l e c u l e does not p o s s e s s a x i a l symmetry. The mag n e t i c e q u i v a l e n c e o f t h e two p i c r y l p r o t o n s , however, s u g g e s t s t h a t t h e p i c r y l r i n g i t s e l f p o s s e s s e s a m o t i o n about t h e N - p i c r y l a x i s , t h e c o r r e l a t i o n t ime f o r the motion b e i n g s h o r t e r than 10 second so t h a t t h e two p r o t o n s appear t o be m a g n e t i c a l l y e q u i v a l e n t i n t h e ENDOR s p e c t r a . The e q u a l l y i n t e n s e q u a r t e t s n e ar 11 and 16 MHz s u p p o r t - t h e a s s i g n m e n t o f t h e DPPH o r t h o p r o t o n s . PAC i s t h e most d i s t o r t e d m o l e c u l e found i n t h i s s t u d y . The e x t r a bond i s h o l d i n g t h e m o l e c u l e i n such a c o n f i g u r a t i o n t h a t t h e p i c r y l r i n g c a n n o t l i e a l o n g any a x i s o f symmetry. The s t e r i c h i n d -r a n c e o f t h e n i t r o groups p r e v e n t s much motion o f the p i c r y l r i n g , and thus t h e c a r b a z y l p o r t i o n o f t h e m o l e c u l e becomes c o m p l e t e l y non-symmetric. T h i s s u r p r i s i n g r e s u l t i s seen o n l y from t h e l o w e r temper-a t u r e ENDOR. F u r t h e r t e m p e r a t u r e v a r i a t i o n on o t h e r m o l e c u l e s e x h i b i t i n g h i g h e r symmetry (such as DPPH) might be i n o r d e r t o d i s c o v e r i f t h e r a p i d motion can be f r o z e n o u t b e f o r e t h e ENDOR enhancement f a l l s t o o low t o be v i s i b l e . 123 4.4 a,Y-Bisdiphenylene-g-phenyl Ally! (BDPA) An interesting analogue to PAC can be found in the stable free radical a,Y-bisdiphenylene-g-phenyl ally! (BDPA) whose structure and numbering scheme are shown in Fig 23. BDPA is the simplest member of a large family of diphenylene ally! stable free radicals which show 88 unusual semiconducting,photoconducting and magnetic properties. 89 In contrast to-PAC, i t possesses a nearly isotropic g-factor, and in contrast to PAC, i t gave ENDOR enhancement with no difficulty at very low microwave powers. The magnetic resonance of BDPA and its analogues has been of interest for several years. In the solid state, only a single exchange-narrowed line is observed in EPR spectra at about 10 GHz and 90 91 24 GHz, from 1.5 K to room temperature. ' Additional lines, corres-9? ponding to g=4 can also be observed in EPR at 20-200 MHz below 2 K. 89 EPR studies at 9.6 GHz indicated that below 6 K linear antiferro-magnetic ordering takes place. This conclusion is supported by both the specific heat 8 9 and magnetic susceptibility data. 9 3 The f i rst investigation of spin delocalization in BDPA was 94 presented by Hausser through EPR studies on solutions. His spectra indicated that in BDPA the unpaired electron has hyperfine coupling with eight equivalent protons, the coupling constant being about 2.2 G. No couplings with the remaining 13 protons were resolved. Subsequent 95 Knight-shift studies by Anderson et a l . also showed the presence of a proton hyperfine couplings although the estimated value of 1.3 G was 94 quite different from that measured by Hausser. 124 0 0 LO —: F i g u r e 23. a , Y - B i s d i p h e n y l e n e - $ - P h e n y l a l l y l f r e e r a d i c a l , showing s t r u c t u r e , numbering scheme and a p p r o x i m a t e c o u p l i n g s ( G ) . 125 A l t h o u g h t h e ENDOR r e s u l t s show t h a t H a u s s e r ' s s t u d y was more c o r r e c t than K n i g h t - s h i f t s t u d i e s , t h e y a l s o r e v e a l t h a t t h e EPR o f Hauss e r i s i n c o m p l e t e and t h a t more c o u p l i n g s a r e n e c e s s a r y than r e p o r t e d t o a c c u r a t e l y d e s c r i b e t h e system. The EPR o f a 10 M s o l u t i o n o f BDPA i n m i n e r a l o i l i s shown i n F i g 2 4 ( a ) . The v e r y narrow s o l u t i o n EPR c o n s i s t s o f seven v i s c o s i t y - b r o a d e n e d l i n e s w i t h a t o t a l s p e c t r a l w i d t h o f l e s s than 20 Gauss. F i g u r e 23 shows why t h i s compound has such a narrow EPR: most o f t h e s p i n d e n s i t y i s on e i g h t c e n t r a l c a r b o n s w i t h no p r o t o n s . The sm a l l amount t h a t d e l o c a l i z e s o f f t h e a l l y ! c h a i n must a l s o be d i v i d e d be-tween two d i p h e n y l e n e r i n g s . Both o f t h e s e c o n d i t i o n s w i l l l e a v e v e r y l i t t l e f o r t h e r i n g p r o t o n s . The ENDOR o f t h e same sample i s shown i n F i g 2 4 ( b ) . E i g h t l i n e s a r e v i s i b l e , a l t h o u g h t h e r e s o l u t i o n o f t h e o u t e r p a i r i s poor. The v a l u e s measured from t h i s spectrum a r e shown i n T a b l e 9. The d i s c r e p a n c y between t h e r e s u l t s o f EPR and t h e K n i g h t -s h i f t measurements a r e now r e s o l v e d : t h e ENDOR r e s u l t s c o n f i r m t h e c o n c l u s i o n s drawn from EPR s t u d i e s . The l a r g e s t p r o t o n h y p e r f i n e c o u p l i n g i s a b o u t 2 Gauss (as i n d i c a t e d by EPR) and not 1.3 Gauss (as i n d i c a t e d by t h e K n i g h t - s h i f t s t u d i e s ) . In f a c t , t h e r e i s no h y p e r f i n e c o u p l i n g o f magnitude 1.3 ± 0.3 Gauss. The ENDOR r e s u l t s a r e a l s o c o n s i s t e n t 96 w i t h t h e EPR r e s u l t s on o t h e r a l l y l r a d i c a l s as s t u d i e s by Haus s e r 97 and by Neugebauer e t a l . The K n i g h t - s h i f t measurements were c a r r i e d o u t a t l i q u i d - h e l i u m t e m p e r a t u r e : s i n c e h y p e r f i n e c o u p l i n g s can v a r y w i t h t e m p e r a t u r e and 126 n i§ 5 n "IS 16 rr F i g u r e 24. (a) EPR o f ( 1 0 _ 3 M ) BDPA i n m i n e r a l o i l as used f o r ENDOR (b) ENDOR o f same BDPA sample a t 340K. TABLE 9 H y p e r f i n e c o u p l i n g s , s p i n d e n s i t i e s and T 9 v a l u e s o b t a i n e d f o r BDPA s o l u t i o n s by ENDOR Atom # Name C o u p l i n g s (Gauss) S p i n D e n s i t i e s 333 K 298 K 1,5,9,13 a l p h a -1.970 + .002 -1. 979 + .002 0.0738 3,7,11,15 gamma -1.886 + .002 -1. 888 + .002 0.0705 2,6,10,14 be t a +0.485 + .002 +0. 489 + .002 -0.0183 4,8,12,16 d e l t a +0.332 + .002 +0. 347 + .002 -0.0129 17,21 19 o r t h o and para phenyl -0. 179 + .004 +0.0067 18.20 meta phenyl +0. 103 + .004 -0.0038 T 2 v a l u e s : ^ 0.42 vs 0, .18 ys (a) QH = -26 .8 G, from low temperature v a l u e s , (b) From ENDOR i n t e n s i t i e s , by computer s i m u l a t i o n 128 s o l v e n t t h e c o u p l i n g s measured by ENDOR need n o t agree w i t h t h o s e d e r i v e d f r o m t h e NMR s t u d i e s . A comparison o f the EPR and NMR r e s u l t s on s i m i l a r f r e e r a d i c a l systems such as DPPH o r pyrene n e g a t i v e i o n shows t h a t t h e l a r g e s t h y p e r f i n e c o u p l i n g s a r e n o t much a f f e c t e d by te m p e r a t u r e . A l l t h e s e o b s e r v a t i o n s s u p p o r t t h e argument by H a m i l t o n 89 e t a l t h a t t h e K n i g h t - s h i f t s i g n a l s may be a s s o c i a t e d w i t h i m p u r i t i e s o r d e f e c t s i n t h e samples used. The ENDOR s p e c t r a show t h a t t h e r e a r e two groups o f p r o t o n s w i t h c o u p l i n g s o f about 2 Gauss.^ S i n c e a t t h i s t e m p e r a t u r e t h e i n t e n s i t i e s o f t h e ENDOR t r a n s i t i o n s c o r r e s p o n d i n g t o a h y p e r f i n e c o u p l i n g o f 2 G a r e e q u a l , t h e e i g h t p r o t o n s must be e q u i v a l e n t i n two groups d f f o u r . F o r more s p e c i f i c a s s i g n m e n t , a c c u r a t e d a t a on m o l e c u l a r s t r u c t u r e o r t h e o r e t i c a l c a l c u l a t i o n s o f h y p e r f i n e i n t e r a c t i o n s i n BDPA would be needed. A c c u r a t e ENDOR d a t a on DPPH and PAC, t o g e t h e r w i t h t h e o r e t i c a l e s t i m a t e s o f h y p e r f i n e c o u p l i n g s used f o r DPPH a r e a v a i l a b l e and can be used. Comparison o f t h e p r e s e n t ENDOR r e s u l t s on BDPA w i t h t h e r e s u l t s on DPPH arid PAC ( T a b l e s 4 and 6) s u g g e s t s t h a t t h e l a r g e s t c o u p l i n g (1.97 G) s h o u l d be a s s i g n e d t o the 4 a l p h a p r o t o n s ( 1 , 5, 9, 13) and t h e next l a r g e s t (1.886 G) t o t h e f o u r gamma p r o t o n s ( 3 , 7, 11, 15). S i m i l a r l y , t h e c o u p l i n g o f -0.5 G; i s a s s i g n e d t o the f o u r b e t a p r o t o n s ( 2 , 6, 10, 14) and t h a t o f 0.33 G t o the f o u r r e m a i n i n g d e l t a p r o t o n s ( 4, 8, 12, 1 6 ) . An i n t e r e s t i n g t e m p e r a t u r e dependence was o b s e r v e d i n the ENDOR s p e c t r a . As the sample was c o o l e d below about 300 K, the i n t e n -s i t i e s o f t h e f o u r o u t e r m o s t l i n e s , b e l o n g i n g t o f o u r a l p h a p r o t o n s and t h e f o u r gamma.' p r o t o n s become anomalous i n t h a t t h e i r r a t i o becomes a p p r o x i -129 m a t e l y 1:2 r a t h e r than 1:1 as e x p e c t e d , s i n c e each o f t h e s e l i n e s a r i s e s from a h y p e r f i n e c o u p l i n g w i t h f o u r p r o t o n s . An ENDOR spectrum showing t h i s anomalous i n t e n s i t y p a t t e r n i s shown i n F i g 25. A comparison w i t h F i g 24 shows t h a t a t low t e m p e r a t u r e s t h e i n t e n s i t i e s o f t h e r e s t o f t h e ENDOR l i n e s a l s o change. The i n t e n s i t i e s o f t h e s e o t h e r l i n e s , however, c o u l d be i n t e r p r e t e d t h r o u g h a p p l i c a t i o n o f t h e T 2 c o r r e c t i o n , w i t h a T 2 - v a l u e i n the range o f 0.1 y s e c a t about 300 K. The t e m p e r a t u r e dependence was r e v e r s i b l e , and a t h i g h e r t e m p e r a t u r e s , t h e i n t e n s i t i e s o f a l l t h e l i n e s can be f i t t e d w i t h a T 2 o f .4 m i c r o -s e c o n d s . T h i s anomaly i n t h e ENDOR i n t e n s i t i e s may be r a t i o n a l i z e d by assuming t h a t t h e tem p e r a t u r e dependence o f t h e r e l a x a t i o n b e h a v i o u r o f t h e f o u r a l p h a p r o t o n s which g i v e r i s e t o t h e a n o m a l o u s - i n t e n s i t y ENDOR l i n e s , i s d i f f e r e n t from t h o s e o f the o t h e r p r o t o n s . T h i s does n o t a g r e e w i t h t h e assumptions t h a t a r e i n v o l v e d i n th e d e r i v a t i o n o f e q q a t i o n 2 3, where T c was assumed (and measured by A l l e n d o e r f e r and M a k i 1 0 ) t o be i d e n t i c a l f o r a l l t h e atoms i n the m o l e c u l e . T h i s assumption h o l d s w e l l f o r a l a r g e number o f systems 18 which show ENDOR enhancement but A t h e r t o n e t a l have a l s o o b s e r v e d c a s e s where i t does n o t . For t he f o u r a l p h a p r o t o n s , i t i s r e a s o n a b l e t o assume t h a t t h e i r r e l a x a t i o n b e h a v i o u r i s d i f f e r e n t from t h a t * o f t h e r e s t o f t h e p r o t o n s s i n c e t h e y a r e the ones n e a r e s t t o the phenyl r i n g . A n m o l e c u l a r model c o n s t r u c t e d f o r BDPA showed t h a t t h e phenyl r i n g must t w i s t o u t o f t h e p l a n e o f t h e r e s t o f t h e m o l e c u l e t o a v o i d s t e r i c h i n d r a n c e from t h e s e 4 a l p h a 131 p r o t o n s . A t h i g h t e m p e r a t u r e s t h e phenyl r i n g might be p e r f o r m i n g h i n d e r e d r o t a t i o n s about t h e C-C bond, p r o v i d i n g some path f o r s p i n -s p i n r e l a x a t i o n f o r t h e p r o t o n s i n the m o l e c u l e . A t lower t e m p e r a t u r e s t h e s e r o t a t i o n s might have s t o p p e d on t h e ENDOR time s c a l e and the phenyl r i n g then would be l o c k e d i n a p o s i t i o n o f minimum s t e r i c hind--, ranee. T h i s would g i v e a d i f f e r e n t e n v i r o n m e n t f o r t h o s e p r o t o n s which come i n c l o s e c o n t a c t w i t h t h e phenyl group. The o u t e r m o s t r i n g p r o t o n s ( a l p h a ) c o u l d thus have a s l i g h t l y d i f f e r e n t r e l a x a t i o n b e h a v i o u r com-p a r e d t o t h e o t h e r p r o t o n s i n t h e BDPA m o l e c u l e . The s l o w i n g down o f th e phenyl r o t a t i o n s h o u l d show up as an i n e q u i v a l e n c e i n t h e o r t h o and meta p r o t o n c o u p l i n g s (as i n the p i c r y l p r o t o n s : o f DPPH). Here t h e sma l l s p i n d e n s i t y on t h e phenyl group and t h e poor i n t e n s i t y o f i t s p r o t o n s i g n a l s r e n d e r t h i s e f f e c t u n o b s e r v a b l e . F i g u r e 26 shows an enl a r g e m e n t o f t h e c e n t r e o f t h e low te m p e r a t u r e s p e c t r u m , and l i n e s due t o the phenyl r i n g a r e v i s i b l e . A d e t a i l e d a s s i g n m e n t i s d i f f i c u l t because t h e s i g n a l - t o - n o i s e o f t h e s e weak s i g n a l s i s low, and a c c u r a t e a p p l i c a t i o n o f t h e T 2 c o r r e c t i o n f o r m u l a depends upon r e l a t i v e l y a c c u r a t e i n t e n s i t i e s . T h i s means t h a t t h e number o f n u c l e i g i v i n g r i s e t o t h e s e l i n e s c a n n o t be d e t e r m i n e d w i t h c e r t a i n t y . The phenyl r i n g has t h r e e ( o r t h o , meta and p a r a ) t y p e s o f p r o t o n s whereas o n l y two ENDOR s i g n a l s a r e e x p e r i m e n t a l l y o b s e r v e d . T h e o r e t i c a l c a l c u l a t i o n s o f h y p e r f i n e c o u p l i n g s o f a s u b s t i t u t e d phenyl r i n g and a comparison w i t h e x p e r i m e n t a l l y o b s e r v e d r e s u l t s on s i m i l a r systems such as t r i p h e n y l m e t h y l J ^ * ^ and V e r d a z y l r a d i c a l s ^ ' ^ 0 0 s u g g e s t t h a t f o r t h e r a t h e r s m a l l s p i n d e n s i t y on t h e phenyl r i n g , t h e n BDPA central lines 132 U F i g u r e 26. En l a r g e m e n t o f c e n t r a l l i n e s o f p r e v i o u s BDPA s p e c t r u m , showing weak phenyl l i n e s . 133 c o u p l i n g s from t h e o r t h o and para p r o t o n s would n o t be r e s o l v e d . The c o u p l i n g s from t h e twonmeta p r o t o n s a r e a l s o e x p e c t e d t o be s m a l l e r by about a f a c t o r o f two compared t o t h o s e f o r t h e o r t h o and the para p r o t o n s . Thus t h e l a r g e r o f t h e s e two s m a l l e s t c o u p l i n g s must be a s s i g n e d t o the t h r e e o r t h o and p a r a p r o t o n s and t h e s m a l l e s t one t o the two meta p r o t o n s . T h i s a s s i g n m e n t p r e d i c t s a r a t i o o f the i n t e n s i t i e s o f t h e s e two s i g n a l s o f 3:2. The o b s e r v e d r a t i o i s about 3:1.5, which i s th o u g h t t o be i n good enough agreement w i t h t h e p r e -d i c t i o n c o n s i d e r i n g t h e s i z e o f the T 2 c o r r e c t i o n n e c e s s a r y f o r t h e s e s m a l l c o u p l i n g s . T h i s i s one c a s e where t h e ENDOR i s n o t c o n c l u s i v e . To check t h i s c o m p l i c a t e d a s s i g n m e n t , EPR s t u d i e s o f BDPA i n t h i n s o l v e n t systems w e r e e s s e n t i a l . A spectrum o f BDPA i n o u t g a s s e d benzene i s shown i n F i g 2 7 ( a ) . The spectrum c o n s i s t s o f n i n e main peaks which have a d d i -t i o n a l , u n r e s o l v e d s t r u c t u r e s on them. The n i n e peaks a r i s e f r o m t h e h y p e r f i n e i n t e r a c t i o n o f t h e e i g h t n e a r l y e q u i v a l e n t a l p h a and gamma p r o t o n s , as was o b s e r v e d by Hausser 9. 6 S i m u l a t i o n o f t h i s - s p e c t r u m w i t h o n l y t h e 4 l a r g e s t c o u p l i n g s o f T a b l e 9 shows i m m e d i a t e l y t h a t i n c l u s i o n o f some phenyl r i n g c o u p l i n g s i s n e c e s s a r y , and s h o u l d have been used on s p e c t r a p u b l i s h e d by H a u s s e t ^ a s w e l l . I n i t i a l s i m u l a t i o n s show s e v e r a l problems a s s o c i a t e d w i t h t h e poor r e s o l u t i o n o f 2 7 ( a ) . I f a l l t h e l i n e s o b s e r v e d i n t h e ENDOR a r e i n c l u d e d , t h e EPR must have 11,250 l i n e s i n 18 Gauss. T h i s i s a lower d e n s i t y o f l i n e s p e r Gauss than o b s e r v e d i n DPPH, but t h e r e a r e few v i s i b l e o v e r l a p s i n t h i s compound. I f t h e spectrum i s s i m u l a t e d w i t h l i n e w i d t h s o f 5 mG.and 70 mG, two more f a c t s a r e o b v i o u s . Even w i t h a s u p e r h e t e r o d y n e 134 F i g u r e 27. (a) EPR o f (10 _ I +M) BDPA i n o u t g a s s e d benzene a t room t e m p e r a t u r e (b): S i m u l a t i o n o f EPR u s i n g ENDOR h y p e r f i n e c o n s t a n t s . 135 s p e c t r o m e t e r , t he t o t a l t h e o r e t i c a l r e s o l u t i o n would not be o b s e r v e d . The h i g h e s t r e s o l u t i o n s t i l l shows a l m o s t a continuum o f l i n e s . Each " l i n e " a t 70 mG c o n s i s t s o f 10 o r more a c t u a l l i n e s . T h i s i m p l i e s t h a t t h e poor r e s o l u t i o n o f 27(a) i s due t o the 100 kHz m o d u l a t i o n l i m i t , and t h a t s u p e r h e t e r o d y n e s p e c t r o s c o p y i s n e c e s s a r y t o prove a b s o l u t e l y the a s s i g n m e n t s f r o m t he EPR. C o u p l i n g s from t h e phenyl p r o t o n s a r e n e c e s s a r y f o r a l l r e a s o n a b l e f i t s o b t a i n e d . A s i m u l a t e d spectrum showing t h e c l o s e s t f i t t o t h e o b s e r v e d spectrum i s shown i n F i g 2 7 ( b ) . The r e s o l u t i o n o f t h e o b s e r v e d spectrum was not adequate t o d i s t i n g u i s h t h e s e two c a s e s : (a) t h e l a r g e r o f t h e two s m a l l e s t c o u p l i n g s '"is from t h e t h r e e o r t h o and para p r o t o n s and t h e s m a l l e s t one i s t h a t from t he meta p r o t o n s , o r (b) t h e s e two c o u p l i n g s a r e from t h e o r t h o and p a r a p r o t o n s , t h a t from t h e meta p r o t o n s b e i n g t o o s m a l l t o be r e s o l v e d by ENDOR. The c o m p a r i s o n m e n t i o n e d above i n d i c a t e s t h a t (a) i s t h e more l i k e l y . The s i m u l a t i o n has few v i s i b l e f e a t u r e s , b ut t o o b t a i n a s h a r p d o u b l e t on t h e c e n t r a l l i n e s , c a s e (b) must be u s e d , and t h e d o u b l e t can a r i s e f r o m t h e s i n g l e p a r a p r o t o n . The s i m u l a t i o n i s shown w i t h c a s e ( b ) , b u t t h e d i f f e r e n c e s a r e v e r y s m a l l . The r e s u l t s on BDPA a r e a l s o c o n s i s t e n t w i t h t h e as s i g n m e n t s f o r PAC and DPPH. In BDPA t h e absence o f ENDOR t r a n s i t i o n s around 15 and 12 MHz c o n f i r m s t h e a s s i g n m e n t o f a c o u p l i n g o f about 1 G t o t h e meta p r o t o n s on t h e p i c r y l r i n g s o f DPPH. S i m i l a r l y t h e p o s i t i o n s o f o t h e r l i n e s , c o r r e s p o n d i n g t o c o u p l i n g s o f magnitudes o f 2 G and 0.5 G a r e c o n s i s t e n t w i t h t h e a s s i g n m e n t f o r DPPH and PAC t o o r t h o ( a l p h a ) and meta ( b e t a ) p r o t o n s r e s p e c t i v e l y . These measurements a l s o r e s o l v e t h e d i s c r e p a n c y 136 between t h e e a r l i e r NMR and EPR r e s u l t s . The ENDOR r e s u l t s a l s o show t h a t on t h e EPR time s c a l e PAC i s more d i s t o r t e d than BDPA. T h i s o b s e r v a t i o n can be q u a l i t a t i v e l y e x p l a i n e d on t h e b a s i s o f t h e m o l e c u l a r s t r u c t u r e s o f PAC and BDPA. The c a r b a z y l r i n g s i n BDPA can d i s t o r t such t h a t t h e phenyl r i n g can s t i l l l i e i n a p l a n e o f symmetry w i t h r e s p e c t t o the two c a r b a z y l r i n g s . F o r the p i c r y l r i n g i n PAC, however, such a p o s i t i o n i s n o t v e r y l i k e l y because o f s t e r i c h i n d r a n c e , as shown by a m o l e c u l a r model o f PAC. The p r e s e n t a s s i g n m e n t i s c o n s i s t e n t w i t h a v a i l a b l e t h e o r e t i c a l and e x p e r i m e n t a l d a t a , but an unambiguous answer must a w a i t ENDOR s t u d i e s on s e l e c t i v e l y d e u t e r a t e d samples. The t e m p e r a t u r e dependence o f t h e i n t e n s i t i e s o f the l a r g e s t c o u p l i n g s i n BDPA a l s o needs f u r t h e r i n v e s -t i g a t i o n . 137 4.5 G e n e r a t e d R a d i c a l s To c o n c l u d e t h i s s t u d y , a number o f l e s s s t a b l e r a d i c a l s were s t u d i e d by ENDOR. The t h r e e i n c l u d e d i n t h i s s e c t i o n a r e p h e n o x a z i n e , p h e n o t h i a z i n e , and 2 - c h l o r o p h e n o t h i a z i n e . As seen i n T a b l e 1 t h e s e a r e a n a l o g u e s o f the s t a b l e r a d i c a l s t h a t have been s t u d i e d . ENDOR s t u d i e s were done t o a s s i s t i n the as s i g n m e n t o f c o u p l i n g s i n the p r e -v i o u s S e c t i o n s , t o t e s t t h e l i n e a r i t y o f the s p e c t r o m e t e r power, and as i d e a l t e s t c a s e s f o r EPR s i m u l a t i o n . A l l o f t h e s e s p e c i e s gave a l m o s t complete r e s o l u t i o n i n t h e i r EPR. T h i s i n i t i a l s t u d y on u n s t a b l e , b i o l o g i c a l l y i n t e r e s t i n g m o l e c u l e s showed a l s o t h a t the s p e c t r o m e t e r c o u l d be used f o r o t h e r t y p e s o f m o l e c u l e s b e s i d e s t h e common s t a b l e f r e e r a d i c a l s . E a r l y work on t h e s e compounds, and o t h e r compounds i n t h i s s e r i e s showed t h a t many d e r i v a t i v e s o f t h e s e compounds s t a b i l i z e d r a d i c a l s . The dye d e r i v a t i v e s o f p h e n o t h i a z i n e have been s t u d i e d by s e v e r a l workers."101-103 j n e promazine and c h l o r o p r o m a z i n e d e r i v a t i v e s have a l s o a t t r a c t e d i n t e r e s t . 1 0 4 " 1 0 6 The v a r i o u s r a d i c a l s t h a t a r e o b t a i n a b l e from p h e n o t h i a z i n e a l o n e have been s t u d i e d with. EPR b y s e v e r a l w o r k e r s . 1 0 7 - 1 1 1 The c o n f u s i o n caused by the many r a d i c a l s t h a t can be o b t a i n e d from p h e n o t h i a z i n e i s a l s o t o be found i n t h e e a r l y l i t e r a t u r e . A s p e c t r u m o f t h e p h e n o t h i a z i n e n e u t r a l r a d i c a l was a s s i g n e d t o both 109 113,114 112 iQg th e p h e n o t h i a z i n e n i t r o x i d e r a d i c a l and the n e u t r a l r a d i c a l . The spectrum o f the n i t r o x i d e was a l s o a s s i g n e d t o both s p e c i e s . T h i s p a r t i c u l a r d i s a g r e e m e n t was e v e n t u a l l y c l a r i f i e d by J a c k s o n and 115 P a t e l u s i n g s p e c i f i c o x i d a n t s such as TTBP, P b 0 2 , KMnO^ and l e a d t e t r a - a c e t a t e . O n l y a few t h e o r e t i c a l c a l c u l a t i o n s 1 1 6 ' 1 1 7 on t h e s e 138 s p e c i e s have been a t t e m p t e d , and t h e s e w i l l be d i s c u s s e d w i t h t he r e s u l t s o b t a i n e d h e r e . 4.5 (a) Phenoxazine P h e n o x a z i n e n e u t r a l r a d i c a l , whose s t r u c t u r e i s shown i n T a b l e 10 was made from t h e p a r e n t p h e n o x a z i n e by hydrogen a b s t r a c t i o n w i t h DPPH. Optimum c o n c e n t r a t i o n s o f around 10 M i n m i n e r a l o i l gave m i c r o -wave, power s a t u r a t i o n w i t h about 25 mW a t room t e m p e r a t u r e . The ENDOR o b t a i n e d i s shown i n F i g 28. T h i s v e r y wide spectrum r e q u i r e d 2 r f bands j on t h e s i g n a l g e n e r a t o r i n use. The l i n e s near 8 and 10 MHz thus have i n t e n s i t i e s t h a t do n o t c o r r e s p o n d t o t h e i n t e n s i t i e s o f t h e o t h e r l i n e s . The l i n e a t 18. MHz i s a l s o near a r e s o n a n c e f r e q u e n c y o f t h e r f t r a n s f o r m e r and a p p e a r s a b n o r m a l l y l a r g e . Assignment was done by comparing t h e measured c o u p l i n g s a g a i n s t t he t h e o r e t i c a l c a l c u l a t i o n s o f C h i n e t a l , ^ ? and by comparison w i t h PAC and BDPA. A l l t h e l i n e s s h o u l d have equal i n t e n s i t y w i t h t h e a s s i g n m e n t s o f T a b l e 9. A 100 kHz EPR spectrum was run i n v e r y w e l l o u t g a s s e d rirheptane and i s shown i n F i g 2 9 ( a ) . T h i s r a d i c a l was a l s o p r e p a r e d by. DPPH b l e a c h -i n g and had a v e r y low l i f e t i m e . In o r d e r t o o b t a i n t h e h i g h r e s o l u t i o n o f 2 9 ( a ) , t h e s o l v e n t s were o u t g a s s e d b e f o r e t h e sample i s added. The o u t e r wing r e s o l u t i o n i s qui t e i m p o r t a n t . A s i m u l a t e d EPR u s i n g o n l y t h e EPR-measured n i t r o g e n c o u p l i n g and t h e ENDOR c o u p l i n g s from m i n e r a l o i l i s shown i n F i g 2 9 ( b ) . The agreement o f t h e i n n e r l i n e s h a p e s and o f t h e wings, i s e x c e l l e n t . S e v e r a l s i m u l a t i o n s w i t h v a r i o u s l i n e w i d t h s were n e c e s s a r y t o o b t a i n such e x a c t agreement. F i g u r e 29. (a) EPR o f (10 - t*M) p h e n o x a z i n e n e u t r a l r a d i c a l i n n-heptane (b) S i m u l a t i o n o f EPR u s i n g ENDOR c o n s t a n t s . 141 The s i m u l a t i o n and t h e e x a c t ENDOR v a l u e s show t h a t t h e u n r e s -t r i c t e d H a r t r e e - F o c k c a l c u l a t i o n s o f r e f e r e n c e 117 f i t t h e o b s e r v e d c o n s t a n t s b e s t . These c a l c u l a t e d s p i n d e n s i t i e s a r e i n c l u d e d i n T a b l e 10 f o r c o mparison. More d e t a i l e d c a l c u l a t i o n s would need e x p e r i m e n t a l c a r b o n and oxygen s p i n d e n s i t i e s b e f o r e much improvement c o u l d be made. 4.5 (b) P h e n o t h i a z i n e The f i r s t o f t h e dye s e r i e s , p h e n o t h i a z i n e was o b t a i n e d as a r a d i c a l i n m i n e r a l o i l by t h e above method. The s o l u b i l i t y o f p h e n o t h i a z i n e i n m i n e r a l o i l i s v e r y low, and t h e l i f e t i m e o f t h e r a d i c a l much l e s s than pheno-t h i a z i n e , about 2 hours. The r a t h e r n o i s y ENDOR o f t h i s r a d i c a l i n m i n e r a l o i l i s shown i n F i g 30. The as s i g n m e n t i s by a n a l o g y t o ph e n o x a z i n e . C o n s i d e r i n g t h e n o i s e , t h e i n t e n s i t i e s a r e c l e a r l y 1:1:1:1, w i t h a v a l u e o f T 2 > .4 m i c r o s e c o n d s . T h e r e a r e no l i n e s near t h e f r e e p r o t o n f r e q u e n c y t o put an upper bound on t h i s v a l u e o f T 2 . The s h o r t l i f e t i m e o f t h i s s p e c i e s p r e v e n t e d use o f both bands of t h e s i g n a l g e n e r a t o r . With t h e c e n t r a l f o u r l i n e s t h e v a l u e o f t h e f r e e p r o t o n f r e q u e n c y c o u l d be checked a g a i n s t t h e measured v a l u e . With t h i s and t h e p r o p e r a p p l i c a t i o n o f e q u a t i o n [ 7 ] t h e c o r r e c t e d c o u p l i n g s from t h e upper l i n e s c o u l d be d e t e r m i n e d . These v a l u e s a r e shown i n T a b l e 10. 115 The v a l u e s measured a g r e e w i t h t h e v a l u e s o f J a c k s o n and P a t e l f o r t h e v a l u e s o b t a i n e d f o r t h e n e u t r a l p h e n o t h i a z i n e r a d i c a l . The a s s i g n -ment was completed by comparison t o ph e n o x a z i n e and by comparing t h e ENDOR v a l u e s t o t h e c a l c u l a t e d v a l u e s o f C h i n e t a l . 1 1 7 P h e n o t h i a z i n e i l l u s t r a t e s some o f t h e d i f f i c u l t i e s w i t h t h e ENDOR t e c h n i q u e . In t h e c a s e o f s h o r t - l i v e d r a d i c a l s i t i s n o t p o s s i b l e F i g u r e 30. Room t e m p e r a t u r e ENDOR o f o u t g a s s e d p h e n o t h i a z i n e n e u t r a l r a d i c a l i n m i n e r a l o i l . T a b l e 10 143 H y p e r f i n e c o u p l i n g s o f g e n e r a t e d r a d i c a l s o b t a i n e d by ENDOR and EPR Phenoxazine P h e n o t h i a z i n e 2 - c h l o r o p h e n o t h i a z i n e a F r o m EPR f i t t i n g . b l v a l u e from r e f 118. o b s e r v e d c a l c u l a t e d N +7.70 ± . 0 1 a + .467 + .490 1 -4.015 ± .002 + .150 + .117 3 -3.056 ± .002 + .114 + .101 4 +0.953 ± .002 -.036 -.042 2 +0.755 ± .002 -.028 -.040 N +7.08 ± .01 a + .429 + .474 1 -3.755 ± .002 + .140 + .116 3 -2.825 ± .002 + .105 + .098 4 +1.002 ± .002 -.037 -.037 2 +0.838 ± .002 -.031 -.037 N +6.90 ± . 0 1 a .418 1 -3.822 ± . 0 0 2 + .143 -8 -3.726 ± . 0 0 2 + .139 -3,6 -2.728 ±.002 + .102 -4,5 +1.004 ±.002 -.036 -7 +0.797 ±.002 -.030 -C l 0.075 ±.01 .050 C -: s o l v e n t : n-heptane I c u l a t i o n s; r e f 117, cQ c-j = -1.5 G, a v e r a g e Phenothiazine b) Simulation 145 t o s c a n t h e e n t i r e ENDOR range b e f o r e t h e sample has n o t i c e a b l y d e c r e a s e d i n c o n c e n t r a t i o n . The low s o l u b i l i t y o f t h e r a d i c a l i n m i n e r a l o i l means t h a t even w i t h a l a r g e sample (13 mm sample tube) t h e a b s o l u t e v a l u e o f t h e enhancement s i g n a l might be v e r y low. For samples w i t h even l e s s s o l u b i l i t y i t would be n e c e s s a r y t o remove t h e t e m p e r a t u r e dewar, and use 25 mm d i a m e t e r samples, i g n o r i n g t h e h e a t i n g e f f e c t from t h e r f f i e l d . The spectrum shown i n F i g 30 was o b t a i n e d o n l y a f t e r 8 optimum s a t u r a t i o n and c o n c e n t r a t i o n samples were i r r a d i a t e d f o r ENDOR. A h i g h r e s o l u t i o n EPR o f 10"1* M p h e n o t h i a z i n e i n n-heptane i s shown i n F i g 3 1 ( a ) . I t i s e a s i e r t o o b t a i n a w e l l r e s o l v e d EPR even w i t h t h i s s h o r t l i v e d sample, as n-heptane can be more c o m p l e t e l y o u t g a s s e d than m i n e r a l o i l , and the wide l i n e s p a c i n g means t h a t s t r o n g e r samples can be used as some c o n c e n t r a t i o n b r o a d e n i n g i s a c c e p t a b l e . A s i m u l a t i o n w i t h t h e measured n i t r o g e n c o u p l i n g and t h e ENDOR v a l u e s i s shown i n F i g 3 1 ( b ) . Good agreement i s v i s i b l e , e s p e c i a l l y on t h e o u t e r f o u r l i n e s o f th e wings. A s l i g h t v a r i a t i o n i n l i n e w i d t h t h r o u g h o u t t h e spectrum p r e v e n t s e x a c t agreement. 4.5 ( c ) C h l o r o p h e h o t h i a z i n e The l a s t member o f t h e s e r i e s , 2 c h l o r o p h e n o t h i a z i n e i s v e r y s o l u b l e i n m i n e r a l o i l , and a s t r o n g sample was p o s s i b l e . I t a l s o had a l i f e t i m e i n m i n e r a l o i l o f o v e r e i g h t h o u r s , so t h a t s l o w e r scans and h i g h e r t i m e c o n s t a n t s on t h e d e t e c t i o n system c o u l d be used. The w e l l r e s o l v e d ENDOR (20 kHz d e v i a t i o n ) o b t a i n e d a t room t e m p e r a t u r e i s shown i n F i g 32. I t i s d i f f i c u l t t o use t h e i n t e n s i t i e s f o r d e t e r m i n i n g t h e number o f p r o t o n s , as a g a i n two s i g n a l - g e n e r a t o r bands had t o be used. Repeated runs a c r o s s t h e b a n d - s w i t c h i n g p o i n t show t h a t i f the 8.5 MHz 3,6 CTl 9 10 l'l ~\2 13 ~~14 15 16 17 F i g u r e 32. ENDOR o f 2 - c h l o r o - p h e n o t h i a z i n e n e u t r a l r a d i c a l i n m i n e r a l 147 d o u b l e t i s from two p r o t o n s , then t h e l i n e a t e x a c t l y 10.0 MHz ( t h e s w i t c h i n g p o i n t ) i s a l s o from two p r o t o n s . S i m i l a r l y , i f t h e o u t e r s e t o f t h e i n n e r l i n e s i s from two p r o t o n s , t h e n t h e i n t e n s i t y r a t i o o f 2:1 p r e d i c t s t h a t t h e i n n e r l i n e can come o n l y from t h e p r o t o n a t p o s i t i o n 7. By a n a l o g y w i t h p h e n o t h i a z i n e , t h e ' o t h e r l i n e s a t 12.2 and 15.2'must come from t h e p r o t o n s a t p o s i t i o n s 4 and 5. Assignment o f t h e o t h e r 1 i n e s i s th e n as shown i n F i g 32. The asymmetry o f t h e m o l e c u l e s p l i t s t h e l a r g e s t c o u p l i n g i n t o two l i n e s near 8.5 MHz. The r e a s o n f o r t h e s i n g l e l i n e a t 10 MHz i s not c l e a r : i t s h o u l d a l s o be s p l i t by the asymmetry as i t i s a s s i g n e d t o Pr o t o n s at. t h e 3"and- 6 p o s i t i o n s . The v a l u e s f o r a l l t h e c o u p l i n g s as measured from t h e ENDOR, th e f r e e p r o t o n v a l u e and e q u a t i o n [7] a r e g i v e n i n T a b l e 10, a l o n g w i t h t h e a s s i g n m e n t s . The EPR o f 2 - c h l o r o p h e n o t h i a z i n e i n o u t g a s s e d n-heptane was e a s i l y o b t a i n e d and i s shown i n F i g 3 3 ( a ) . The f i r s t s i m u l a t i o n s o f t h e EPR from the ENDOR v a l u e s d i d not r e p r o d u c e e x a c t l y t h e EPR, e s p e c i a l l y t h e o u t e r l i n e s . An en l a r g e m e n t o f t h e o u t e r l i n e s i s shown i n F i g 3 4 ( a ) . The above a s s i g n m e n t does not g i v e enough s h o u l d e r s and s p l i t t i n g s t o t h e s e o u t e r l i n e s , and i s shown i n F i g 3 4 ( c ) . The c o n s t a n t s and t h e above a s s i g n m e n t s i m u l a t e e x a c t l y t h e ENDOR spectrum i f a v a l u e of T 2 = 2.7 x 1 0 " 7 sec i s used and t h e l i n e s below 10 MHz a r e c o r r e c t e d f o r t h e d i f f e r e n t fm d e v i a t i o n . In an at t e m p t t o o b t a i n a c l o s e r f i t , s e v e r a l EPR s p e c t r a ' were s i m u l a t e d w i t h s m a l l d i f f e r e n c e s i n t h e 4 and 5 p o s i t i o n c o u p l i n g . These two p o s i t i o n s c o u l d be e x p e c t e d t o be d i f f e r e n t on symmetry a l o n e , but none.of t h e s i m u l a t i o n s c - t r i e d r e p r o -duced t h e o u t e r l i n e p a t t e r n . 148 (b) Simulation F i g u r e 33. (a) EPR o f c h l o r o - p h e n o t h i a z i n e n e u t r a l r a d i c a l i n n-heptane a t room t e m p e r a t u r e and (b) S i m u l a t i o n o f EPR u s i n g ENDOR c o n s t a n t s , 149 F i n a l l y , a s m a l l c h l o r i n e c o u p l i n g was used i n t h e s i m u l a t i o n . A range o f p o s s i b l e c h l o r i n e v a l u e s was s e l e c t e d , and many s i m u l a t i o n s o f t h e o u t e r l i n e s were r u n w i t h t h e ENDOR p r o t o n c o n s t a n t s , and v a r i o u s c h l o r i n e v a l u e s . (The program QUICK can be used t o s i m u l a t e c o u p l i n g s w i t h s p i n I = 3/2 by o v e r l a p p i n g c o u p l i n g s o f I = 1/2). The c l o s e s t f i t was o b t a i n e d w i t h = 0.075 G and i s shown i n F i g 3 4 ( b ) . T h i s v a l u e gave s h o u l d e r s on t h e c o r r e c t s i d e o f t h e l a r g e l i n e s as shown. A s i m u l a -t i o n o f t h e e n t i r e EPR i s shown i n F i g 3 3 ( b ) , u s i n g t h e s e v a l u e s . I t i s not c l e a r f r o m t h e s i m u l a t i o n o f F i g 34(b) t h a t a q u a r t e t f r o m c h l o r i n e has been added: t h i s i s v i s i b l e o n l y when t h e l i n e w i d t h i s lowered t o 25 mG. T h i s c o u p l i n g f o r c h l o r i n e i s v e r y c l o s e t o t h e 100 kHz EPR m o d u l a t i o n f r e q u e n c y l i m i t f o r r e s o l u t i o n . T h i s i s one o f t h e few i n s t a n c e s o f a c h l o r i n e c o u p l i n g i n s o l u t i o n s p e c t r a l ^ I f o t h e r p l a n a r a r o m a t i c r a d i c a l s ^ have c o u p l i n g s i n the same r a n g e , i t i s c l e a r why t h e y have been .observed so r a r e l y . I f a c o u p l i n g i s equal t o o r l e s s than t h e l i n e w i d t h l i m i t , i t i s not o b s e r v e d as a symmetric l i n e p a t t e r n , but j u s t broadens and o v e r l a p s t h e l i n e s . T h i s example shows t h e advantage o f t h e ENDOR t e c h n i q u e . As w i t h DPPH, s m a l l , d i f f i c u l t - t o - m e a s u r e c o u p l i n g s can be o b t a i n e d by v a r i a t i o n o f pos-s i b l e - v a l u e s i f t h e p r o t o n c o u p l i n g s a r e known. I n s t e a d o f comparing a l l o f t h e v a l u e s f r o m t h e EPR, i t i s s a t i s f a c t o r y t o use t h e ENDOR-obtained-v a l u e s o n l y and v a r y o n l y t h e unknown c o u p l i n g ( i n t h i s c a s e t h e C l ) . The g e n e r a t e d r a d i c a l s and t h e i r f i t t e d EPR show c l e a r l y t h a t the ENDOR i n m i n e r a l o i l g i v e s v a l u e s v e r y c l o s e t o t h o s e o f EPR i n n-heptane. T h i s s t r e n g t h e n s t h e argument f o r t h e ass i g n m e n t o f DPPH and PAC. The ENDOR v a l u e s s h o u l d be v e r y c l o s e t o t h e EPR v a l u e s , and t h e parameters o b t a i n e d Cl-Phenothiazine Outer Lines (a) Spectrum Simulation with Cl Simulation without Cl F i g u r e 34. (a) E n l a r g e m e n t o f o u t e r l i n e s o f c h l o r o - p h e n o t h i a z i n e (b) S i m u l a -t i o n s o f o/uter l i n e s w i t h c h l o r i n e c o u p l i n g (aci = 75 mG) and ( c ) S i m u l a t i o n o f o u t e r l i n e s w i t h o u t c h l o r i n e c o u p l i n g . 151 by o t h e r t e c h n i q u e s s h o u l d s i m u l a t e both EPR and ENDOR s p e c t r a . I t c o u l d be arg u e d t h a t t h e d i f f e r e n c e v i s i b l e i n F i g 17 i s m e r e l y a s o l v e n t s h i f t from m i n e r a l o i l t o n-heptane, but as shown by t h e s e l a s t samples, q u i t e c l o s e agreement c an be o b t a i n e d i n m i n e r a l o i l . A f u r t h e r s t u d y on t h e ENDOR o f o t h e r members o f t h i s s e r i e s o f dyes c o u l d now be done, as t h e i n i t i a l compounds appear t o g i v e good s i m u l a t i o n s o f t h e i r EPR. The h i g h e r members o f t h e s e r i e s have no r e s o l u t i o n e x c e p t f o r a 5 or 7 broad-1 i n e s p e c t r u m , so t h a t t h e ENDOR v a l u e s must be used e n t i r e l y . 152 REFERENCES 1. 6. F e h e r , Phys. Rev. 103, 834 (1956). 2. See N.S. D a l a i , Ph.D. T h e s i s , U.B.C., f o r work h e r e ; and A.L. Kwiram i n Annual Reviews o f Phys. Chem. 22, 133 (1971); and A. Abragam and B. 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