NMR Spectroscopy: New Methods and Applications 9780841207233, 9780841208926, 0-8412-0723-2

Table of contents :
Title Page ......Page 1
Half Title Page ......Page 3
Copyright ......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
1 Introduction......Page 8
LITERATURE CITED......Page 12
2 Ultra High-Field NMR......Page 13
Magnet Technology......Page 15
Instrumental Requirements at Very High Field......Page 20
Typical Applications......Page 25
LITERATURE CITED......Page 35
3 NMR Spectroscopy at 600 MHz......Page 36
Literature Cited......Page 49
4 The Information Content of Two-Dimensional Fourier Spectroscopy......Page 51
LITERATURE CITED......Page 64
5 Quadrupolar Metallic Nuclei 23Na NMR Studies of Cation Binding by Natural and Synthetic Ionophores......Page 66
Theoretical Background: Dual Relaxation of a Spin 3/2 Particle......Page 67
Experimental Procedure and Data Analysis......Page 69
Application to Structure Reactivity Correlations......Page 73
Site Binding or Atmospheric Condensation Polyelectrolytes......Page 74
Results for Ionophore Antibiotics......Page 79
Cation-Binding Muscular Proteins......Page 82
Structures Maintained by Na+- Binding: The 5+-GMP Supramolecular Assembly......Page 88
23Na NMR Whole Tissue......Page 91
LITERATURE CITED......Page 93
Magnetic Properties of Deuterium......Page 99
High Resolution Deuterium NMR......Page 102
Deuterium Nuclear Relaxation......Page 108
Wide-line Deuterium NMR......Page 111
References......Page 118
Spectral Characteristics of 13C Enriched Amino Acids......Page 120
Conformational Interpretation of Scalar Carbon-Carbon Coupling Constants.......Page 128
Relaxation Behavior of 13C Enriched Amino Acids.......Page 139
Biological Studies with Istopically Labeled Amino Acids and Peptides......Page 149
Future Developments......Page 151
LITERATURE CITED.......Page 152
8 The Study of the Metabolism of 13C Labeled Substrates by 13C NMR Spectroscopy of Intact Cells, Tissues, and Organs......Page 157
Methodology......Page 160
Applications......Page 171
Prospects for Future Developments......Page 182
Literature Cited......Page 183
9 Chemical Bond Labeling and Double Cross-Polarization NMR......Page 187
NMR Apparatus......Page 188
Specific Double Labels: Soybean Organ Cultures......Page 190
Non-Specific Double Labels: Protein Turnover in Soybean Leaves......Page 195
Conclusions and Projections......Page 197
Literature Cited......Page 198
Multinuclear High Resolution FT NMR at High Pressure......Page 199
Relaxation and Transport Behavior of Compressed Supercritical Water......Page 207
Dynamic Structure of Disordered Materials......Page 209
Literature Cited......Page 217
11 13C Cross-Polarization Magic-Angle Spinning NMR Study Amines Adsorbed on γ-Alumina Utilizing a Unique Spinner Design......Page 218
Experimental......Page 219
The 13C NMR of Amines on γ-Alumina......Page 223
Conclusions and Prospects for the Future Research......Page 227
LITERATURE CITED......Page 228
12 Solid State NMR of Linear and Cyclic Peptides......Page 231
Conformational Analysis of Isotropic Chemical Shifts......Page 232
Dynamical Analysis of Lineshapes......Page 237
Conformational Analysis of 14N-13C Dipolar Couplings......Page 239
Literature Cited......Page 244
13 31P NMR Studies of DNA Conformation and Dynamics......Page 246
Allernating B-DNA......Page 247
Dynamics ......Page 257
Telestability......Page 260
Acknowledgement......Page 262
Literature Cited......Page 263
14 Multiple Field Natural Abundance 13C NMR Studies of DNA Dynamics......Page 265
Literature Cited......Page 278
15 Photochemically Induced Dynamic Nuclear Polarization (Photo-CIDNP) of Biological Molecules Using Continuous Wave and Time-Resolved Methods......Page 280
The Radical Pair Mechanism of CIDNP......Page 281
Cyclic Reactions and Cancellation......Page 285
Photo CIDNP of Biological Molecules......Page 298
Time Resolved Photo-CIDNP......Page 307
Literature cited......Page 312
16 13C NMR Characterization of Solid Fossil Fuels Using Cross-Polarization and Magic-Angle Spinning......Page 314
Coals......Page 318
Oil Shales......Page 322
Structural Resolution......Page 331
Summary and Conclusions......Page 335
References and Footnotes......Page 337
Solid State 13C NMR Studies of Polyester Thermoplastic Elastomers......Page 339
Experimental Section......Page 340
Results — Mobile Domains......Page 342
Results — Rigid Domains......Page 347
Discussion......Page 351
Literature Cited......Page 353
A......Page 355
B ......Page 356
C ......Page 357
D ......Page 362
F ......Page 365
G ......Page 366
H ......Page 367
I ......Page 368
L ......Page 369
M ......Page 370
N ......Page 372
P ......Page 373
R ......Page 376
S ......Page 377
T ......Page 380
V ......Page 381
Z ......Page 382

Citation preview

N M R Spectroscopy: New Methods and Applications

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

N M R Spectroscopy: New Methods and Applications George C. Levy, EDITOR Syracuse University

Base

symposiu

jointly

sponsored by the Divisions of Analytical, Organic, and Physical Chemistry at the 181st Meeting of the American Chemical Society, Atlanta, Georgia, March 29-April 3, 1981.

ACS SYMPOSIUM SΕRIΕS 191

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1982

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Library of Congress Cataloging in Publication Data NMR spectroscopy. (ACS symposium series, ISS "Based on a symposium jointly sponsored by e Division [s] of Analytical, Organic, and Physical Chemistry at the 181st meeting of the American Chemical Society, Atlanta, Ga., March 29-April 3, 1981." Includes index. 1. Nuclear magnetic resonance spectroscopy—Congresses. I. Levy, George C. II. American Chemical Society. Division of Analytical Chemistry. III. American Chemical Society. Division of Organic Chemistry. IV. American Chemical Society. Division of Physical Chemistry. V . Title: NMR spectroscopy. VI. Series. QD96.N8N59 1982 543.0877 82-11458 ISBN 0-8412-0723-2 ACSMC8 191 1-388 1982

Copyright © 1982 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED

STATES

OF

AMERICA

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

A C S Symposium Series M . Joan Comstock, Series Editor

Advisory Board David L. Allara

Marvin Margoshes

Robert Baker

Robert Ory

Donald D. Dollberg

Leon Petrakis

Robert E. Feeney

Theodore Provder

Brian M. Harney

Charles N. Satterfield

W. Jeffrey Howe

Dennis Schuetzle

James D. Idol, Jr.

Davis L. Temple, Jr.

Herbert D. Kaesz

Gunter Zweig

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

FOREWORD The ACS S Y M P O S I U M SERIES was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S I N C H E M I S T R Y SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

PREFACE T H E

PAST T E N YEARS HAVE S E E N

P H E N O M E N A L D E V E L O P M E N T S in

the

methodology and application of nuclear magnetic resonance (NMR) to chemistry and biophysics. The introduction of Fourier transform N M R instrumentation was quickly followed by major extensions in instrumental capabilities. As a result, the two chief characteristics of N M R spectra, sensitivity and resolution, underwent radical improvements. Applications once essentially restricted to a few nuclei became established broadly across the entire periodi of very complex systems-including proteins, nucleic acids, and crystalline and amorphous solids-are now common. Other inventions, like multidimensional F T NMR, N M R imaging, and multiple quantum N M R spectroscopy, encourage future developments. These chapters describe many current advances in N M R spectroscopy. Most are reports based on lectures given in April 1981. The Editor would like to thank all of the authors for their exciting presentations. G E O R G E C.

LEVY

Syracuse University Syracuse, New York February 1, 1982

ix

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

1 Introduction GEORGE C. LEVY and DAVID J. CRAIK Syracuse University, Department of Chemistry, Syracuse, NY 13210

Nuclear Magnetic Resonance (NMR) Spectroscopy has developed into an ever expanding in its 35 year history, spectroscopy adapte use by physicists, who had first discovered it, to the realm of chemists who saw the potential of the so-called "chemical shift" phenomenon as a structural probe. This first useful parameter has now been supplemented by many other experimentally accessible quantities, as well as a labyrinth of new applications and methods. In this monograph, we hope to outline some of these new and exciting developments in NMR spectroscopy. Most of the chapters are based on symposium lectures given at the 181st National Meeting of The American Chemical Society, in Atlanta Georgia, March, 1981. Several additional manuscripts were solicited from leading NMR spectroscopists. In broad terms, most new applications of NMR in recent years have derived from parallel improvements in instrumentation and methods. The instrumental improvements may be categorized as follows: 1.

The development and use of spectrometers operating at higher magnetic f i e l d s , i n some cases with large and versatile probe (sample) geometries. 2. The development of multi-nuclear spectrometers. 3. Improved spectrometer design for Fourier Transform techniques; higher sensitivity for proton ( H) NMR and a l l other nuclei. 4. Advances in computer capabilities. Magnetic f i e l d strength. Since the early 1970 s there has been increasing use of superconducting solenoid based systems which are capable of extremely high magnetic fields. The highest f i e l d s i n use today (11.7-14.4 Tesla) correspond to proton NMR frequencies of 500-600 MHz. The development of wide-bore superconducting magnets also represents a s i g n i f i c a n t instrumental improvement that has made possible many new applications. The f i r s t important benefit i s an increased sample 1

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0097-6156/82/0191-0001$06.00/0 © 1982 A m e r i c a n Chemical Society

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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volume and thus i n c r e a s e d s e n s i t i v i t y : the l a r g e r the number o f n u c l e i w i t h i n the r e c e i v e r c o i l the stronger the s i g n a l . S e c o n d l y , a l a r g e r probe v o l u m e a l l o w s more f l e x i b i l i t y w i t h r e s p e c t t o t h e n a t u r e and g e o m e t r y o f t h e e x p e r i m e n t . For e x a m p l e , i t i s now p o s s i b l e t o p l a c e o r g a n s o r even l i v i n g animals i n t o the probe o f a h i g h r e s o l u t i o n magnet. M u l t i - n u c l e a r NMR. I t was o f c o u r s e no a c c i d e n t t h a t t h e e a r l i e s t n u c l e i s t u d i e d by NMR w e r e t h o s e w i t h t h e h i g h e s t s u s c e p t i b i l i t y t o d e t e c t i o n . NMR s t u d i e s o f l e s s a c c e s s i b l e n u c l e i awaited improvements i n instrument s e n s i t i v i t y , and i t was n o t u n t i l t h e 1 970's t h a t m u l t i - n u c l e a r s p e c t r o m e t e r s d r a m a t i c a l l y expanded the range o f p o t e n t i a l a p p l i c a t i o n s o f NMR spectroscopy. NMR i s no longer o n l y a s s o c i a t e d w i t h a few n u c l e i such as 'H, '3c and ^^F, b u t has a l s o been a p p l i e d t o many others, i n c l u d i n g H, N , 0 , N a , Cd, P t t o name a few. F o u r i e r Transfor been improved spectromete t e c h n i q u e o f p u l s e d F o u r i e r t r a n s f o r m NMR was c o m m e r c i a l i z e d around 1970 and b r o u g h t an i m m e d i a t e 10-15 f o l d i n c r e a s e i n s e n s i t i v i t y f o r C NMR (as w e l l as a s m a l l e r f a c t o r observed f o r 'H NMR). I t i s l e s s w e l l known, b u t d u r i n g t h e l a s t 10 y e a r s , i m p r o v e m e n t s i n s p e c t r o m e t e r d e s i g n (e.g. o p t i m i z a t i o n o f r e c e i v e r c o i l and e l e c t r o n i c c i r c u i t s f o r pulsed operation) have p r o d u c e d an a d d i t i o n a l o r d e r o f m a g n i t u d e g a i n i n s e n s i t i v i t y , e s s e n t i a l l y matching the i n i t i a l advantage o f FT NMR. Computer developments. Increased computer c a p a b i l i t i e s have a l s o g r e a t l y c o n t r i b u t e d t o new a p p l i c a t i o n s . The modern NMR computer system and s o f t w a r e can c o n t r o l complicated m u l t i p u l s e e x p e r i m e n t s where many f a c t o r s such as d e l a y t i m e s , d e c o u p l e r l e v e l s , p u l s e w i d t h s o r r f phase may be v a r i e d s y s t e m a t i c a l l y . I n f a c t , v i r t u a l l y a l l spectrometer f u n c t i o n s a r e now s e t under computer c o n t r o l . The second f a c t o r r e s p o n s i b l e f o r many recent a p p l i c a t i o n s i s t h a t o f c o n c e p t u a l l y new NMR methods. Some o f t h e s e a r e l i s t e d below: 1. Two-dimensional F o u r i e r Transform NMR. 2. High r e s o l u t i o n NMR i n s o l i d s . 3· New k i n d s o f p u l s e sequences. 4. Chemically Induced Dynamic Nuclear P o l a r i z a t i o n (CIDNP) 5. M u l t i p l e Quantum NMR. 6. NMR imaging. T h i s g e n e r a l breakdown r e p r e s e n t s a somewhat a r b i t r a r y d i v i s i o n o f s u b j e c t s and t e c h n i q u e s . I n f a c t , t h e r e i s a g r e a t d e a l o f o v e r l a p among t h e v a r i o u s c a t e g o r i e s , as w i l l be seen f r o m t h e d i s c u s s i o n b e l o w , and f r o m t h e r e m a i n i n g c h a p t e r s o f t h i s monograph. Two-dimensional £1 M l spectroscopy. The e x c i t i n g new techniques o f 2D FT NMR spectroscopy a r e c u r r e n t l y being a p p l i e d t o a v a r i e t y o f c h e m i c a l and b i o l o g i c a l p r o b l e m s . 2D FT NMR i n v o l v e s the c o l l e c t i o n o f data as a f u n c t i o n o f two independent 2

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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t i m e d o m a i n s , t ^ and t f o l l o w e d by a d o u b l e F o u r i e r t r a n s f o r m a t i o n . The r e s u l t a n t 2D spectrum c o n t a i n s one i n t e n s i t y a x i s and two frequency axes. A l a r g e v a r i e t y o f 2D experiments a r e p o s s i b l e , d e p e n d i n g on t h e p e r t u r b a t i o n s (e.g. f r e q u e n c y o r phase o f r f i r r a d i a t i o n , decoupler l e v e l , etc.) t h a t a r e a p p l i e d to the nuclear s p i n s d u r i n g the i n t e r v a l s t-j and t . Most but not a l l a p p l i c a t i o n s o f 2D c o r r e l a t e d spectroscopy to date have i n v o l v e d C and H s t u d i e s . A 2D spectrum i n which one a x i s r e p r e s e n t s C chemical s h i f t s and the other *E chemical s h i f t s y i e l d s c r o s s peaks o n l y f o r coupled n u c l e i ( i . e . there i s a d e g r e e o f c o r r e l a t i o n between t h e " c and 'H s p e c t r a ) . T h i s i n f o r m a t i o n o f t e n a l l o w s complete assignments t o be made i n the i n d i v i d u a l C o r Η s p e c t r a o f complex organic molecules. W h i l e 2D c o r r e l a t e d spectroscopy p r o v i d e s i n f o r m a t i o n which i s not d i r e c t l y o b t a i n a b l fro 1D experiment anothe c l a s f 2D e x p e r i m e n t s , c o l l e c t i v e l s i m p l i f i e s complex s p e c t r y spreading spectrum i n t o a second dimension. T y p i c a l spreading parameters i n c l u d e s c a l a r c o u p l i n g s , d i p o l a r c o u p l i n g s , o r chemical s h i f t s . When s c a l a r c o u p l i n g c o n s t a n t s a r e used a s t h e s p r e a d i n g parameter the technique i s c a l l e d J - r e s o l v e d 2D spectroscopy, and c h e m i c a l s h i f t and c o u p l i n g i n f o r m a t i o n c a n be e f f e c t i v e l y separated. This i s a very powerful and i m p o r t a n t f e a t u r e o f 2D FT NMR s p e c t r o s c o p y , a s i t i s o f t e n v e r y d i f f i c u l t t o o b t a i n c o u p l i n g constants from normal (1D) s p e c t r a o f complex m o l e c u l a r systems because o f the o v e r l a p o f many m u l t i p l e t s . Spreading the o v e r l a p p i n g peaks i n complex H spectrum i n t o a second dimension o f t e n g r e a t l y s i m p l i f i e s s p e c t r a and a l l o w s c o m p l e t e peak a s s i g n m e n t s t o be made. U s i n g a p p r o p r i a t e p r o j e c t i o n s i n t h i s t y p e o f J - r e s o l v e d 2D p r o t o n s p e c t r u m i t i s i n f a c t p o s s i b l e t o p r o d u c e a s i m u l a t e d broadband h o m o n u c l e a r d e c o u p l e d proton spectrum. NMR s p e c t r o s c o p y o f s o l i d s a m p l e s . The d e v e l o p m e n t o f methodology t o o b t a i n h i g h r e s o l u t i o n s p e c t r a o f s o l i d s has g r e a t l y enhanced t h e r a n g e o f p o t e n t i a l NMR a p p l i c a t i o n s , p a r t i c u l a r l y f o r s t u d i e s o f low n a t u r a l abundance n u c l e i such as "c. Under the normal c o n d i t i o n s used t o o b t a i n ' C NMR s p e c t r a o f l i q u i d s , a s o l i d s a m p l e w o u l d y i e l d o n l y an e x t r e m e l y b r o a d f e a t u r e l e s s spectrum. A l a r g e c o n t r i b u t i o n t o the broadening a r i s e s from s t a t i c d i p o l a r i n t e r a c t i o n s . ' C s p e c t r a o f s o l i d s o b t a i n e d u s i n g h i g h power p r o t o n i r r a d i a t i o n ( o f t e n t e r m e d d i p o l a r d e c o u p l i n g ) c a n have much o f t h e i n i t i a l broadening removed, but s t i l l may have l i n e w i d t h s o f 5-10 KHz o r more. This b r o a d e n i n g i s due t o c h e m i c a l s h i f t a n i s o t r o p y . The o b s e r v e d b r o a d e n v e l o p e i s a r e s u l t o f c o n t r i b u t i o n s f r o m t h e many i n d i v i d u a l chemical s h i f t s of n u c l e i i n molecules oriented d i f f e r e n t l y w i t h i n the sample. The a n i s o t r o p y can be e f f e c t i v e l y removed i f t h e s a m p l e i s spun r a p i d l y a t an a n g l e o f 54.7° ( t h e magic .angle) w i t h r e s p e c t t o the e x t e r n a l magnetic f i e l d . 2

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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Using the techniques o f d i p o l a r decoupling and magic angle s p i n n i n g i t i s p o s s i b l e t o produce s p e c t r a i n s o l i d s h a v i n g n e a r l y the same r e s o l u t i o n as i s obtained i n l i q u i d s . However, t h e l o w n a t u r a l abundance o f C n u c l e i , and t h e i r l o n g e r 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 s (T^s) i n s o l i d s compared w i t h l i q u i d s s e v e r e l y l i m i t s the s i g n a l - t o - n o i s e r a t i o t h a t can be obtained i n a g i v e n time f o r a s o l i d sample. This s e n s i t i v i t y problem can be overcome u s i n g the t e c h n i q u e o f c r o s s p o l a r i z a t i o n (CP), w h i c h not o n l y b r i n g s about an i n c r e a s e i n s e n s i t i v i t y by a l l o w i n g the m a g n e t i z a t i o n from the abundant H n u c l e a r s p i n s t o be t r a n s f e r r e d ( c r o s s p o l a r i z e d ) to the d i l u t e C n u c l e i , but a l s o a l l o w s s i g n a l accumulation to be repeated a t i n t e r v a l s r e l a t e d to the s h o r t e r H r e l a x a t i o n t i m e s r a t h e r t h a n t h e l o n g e r ' C r e l a x a t i o n times. I n f a c t , i t i s a l s o u s u a l t o p e r f o r m the u n r e l a t e d MAS experiment i n c o n j u n c t i o n w i t h c r o s s p o l a r i z a t i o n (these are s p e c i f i e d a demonstrates the remarkabl t h e - a r t C CPMAS spectrum of s o l i d r e s e r p i n e . ff¥lti-puJ.$e t e c h n i q u e s . Today, the s i m p l e r e p e t i t i v e s i n g l e p u l s e FT NMR e x p e r i m e n t has been augmented by p u l s e schemes designed to probe d i f f e r e n t parameters or phenomena or to improve s e n s i t i v i t y . One new sequence, which i s c u r r e n t l y g a i n i n g w i d e s p r e a d a p p l i c a t i o n h a s b e e n g i v e n t h e acronym INEPT ( I n s e n s i t i v e N u c l e i Enhanced by P o l a r i z a t i o n T r a n s f e r ) . ( 2 ) I t provides s i g n i f i c a n t s i g n a l enhancements, p a r t i c u l a r l y f o r n u c l e i such as N o r ^ S i but a l s o p r o v i d e s a means o f d e t e r m i n i n g s i g n a l m u l t i p l i c i t y i n proton decoupled C s p e c t r a . C h e m i c a l l y I n d u c e d Dynamic N u c l e a r P o l a r i z a t i o n (CIDNP). T h i s t e r m has been used t o d e s c r i b e t h e enhancement o f n u c l e a r s p i n p o l a r i z a t i o n o b s e r v e d i n the NMR s p e c t r a o f compounds u n d e r g o i n g r a d i c a l r e a c t i o n s . Some e x c i t i n g a p p l i c a t i o n s a r e d e s c r i b e d i n Chapter X. M u l t i p l e quantum NMR The s i g n a l s o b s e r v e d i n n o r m a l NMR s p e c t r a a r i s e f r o m t r a n s i t i o n s w h i c h obey the s e l e c t i o n r u l e Am=±.1 (m i s t h e t o t a l m a g n e t i c quantum number o f t h e s p i n system). Using s p e c i a l pulse techniques i t i s p o s s i b l e t o e x c i t e m u l t i p l e quantum "coherences* between s t a t e s where m=±2,3 etc. A l t h o u g h not d i r e c t l y o b s e r v a b l e t h e s e c o h e r e n c e s can be converted, u s i n g s p e c i a l pulse techniques, i n t o s i g n a l s which can be detected. I n the l a s t few years, s t u d i e s o f m u l t i p l e quantum t r a n s i t i o n s i 3 l have been c a r r i e d out i n l i q u i d s , l i q u i d c r y s t a l s and s o l i d s , and promise t o provide much i n f o r m a t i o n on m o l e c u l a r s t r u c t u r e s , conformations and c o r r e l a t e d motions. Specifically, the r e l a t i v e s i m p l i c i t y of m u l t i p l e quantum s p e c t r a g r e a t l y a i d s i n s t r u c t u r a l determinations of molecules aligned i n l i q u i d c r y s t a l s . I n a d d i t i o n , r e l a x a t i o n measurements o b t a i n e d f r o m m u l t i p l e quantum s p e c t r a are p a r t i c u l a r l y v a l u a b l e i n d e t e r m i n i n g complex a n i s o t r o p i c or c o r r e l a t e d m o l e c u l a r m o t i o n s . Multiple quantum methods a l s o p r o m i s e t o be e x t r e m e l y u s e f u l f o r assignment purposes. For example, i t has been r e c e n t l y shownULi. 3

3

1

3

3

1 5

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

1.

5

Introduction

L E V Y A N D CRAIK

t h a t a combination o f the p o w e r f u l methods o f 2D spectroscopy and d o u b l e quantum NMR a l l o w s C - C c o u p l i n g c o n s t a n t s t o be r e a d i l y measured and a s s i g n e d i n n a t u r a l abundance s a m p l e s . Using t h i s c o u p l i n g i n f o r m a t i o n i t i s p o s s i b l e t o s y s t e m a t i c a l l y b u i l d up a p i c t u r e o f t h e c o n n e c t i v i t y o f c a r b o n atoms i n a molecule and hence r e a d i l y determine the e n t i r e s t r u c t u r e o f the carbon skeleton. NMR imaging. This d e s c r i b e s a c l a s s o f experiments i n which NMR s i g n a l s a r e used t o o b t a i n i n f o r m a t i o n having s p a t i a l s i g n i f i c a n c e . Most imaging experiments i n v o l v e the d e t e c t i o n o f proton s i g n a l s , although some e x p e r i m e n t s have been done u s i n g other h i g h l y s e n s i t i v e n u c l e i such as '^F. ^he i n s t r u m e n t s used i n these experiments may be q u i t e d i f f e r e n t from standard h i g h r e s o l u t i o n NMR s p e c t r o m e t e r s , b u t t h e y o p e r a t e on t h e same p r i n c i p l e : n u c l e i having a magnetic moment absorb r f energy a t a frequency d i r e c t l y p r o p o r t i o n a field. I n the imaging L a u t e r b u r i S l a f i e l d g r a d i e n t i s superimposed on the main f i e l d so t h a t d i f f e r e n t p a r t s o f t h e s a m p l e e x p e r i e n c e d i f f e r e n t magnetic f i e l d s and hence resonate a t d i f f e r e n t f r e q u e n c i e s . The r e s u l t a n t NMR s i g n a l s p r o v i d e a l i n e a r p r o f i l e o f t h e d i s t r i b u t i o n o f m a g n e t i c n u c l e i a c r o s s t h e s a m p l e , and when f u r t h e r scans a r e taken, u s i n g g r a d i e n t s i n d i f f e r e n t d i r e c t i o n s , i t i s p o s s i b l e t o r e c o n s t r u c t a two o r three d i m e n s i o n a l image o f the sample. Recent d e v e l o p m e n t s i n f i e l d g r a d i e n t and o t h e r imaging methods a r e d i s c u s s e d i n some d e t a i l i n a r e c e n t review by Hoult^Êl Methods which do not u t i l i z e a s t a t i c l i n e a r f i e l d g r a d i e n t , f o r example t h e " f i e l d f o c u s s i n g " t e c h n i q u e o f DamadianJjQ. a r e a l s o i n c u r r e n t use. I n t h i s t e c h n i q u e and i n r e l a t e d methods i t i s p o s s i b l e t o shape t h e m a g n e t i c f i e l d i n such a manner as t o focus on s p e c i f i c volumes w i t h i n the sample. 1 3

3

LITERATURE CITED 1. Levy, G. C.; Lichter, R. L.; Nelson, G. L. Carbon-13 Nuclear Magnetic Resonance Spectroscopy, (2nd edition) Chapter 10, Wiley, New York 1980. 2. a) Morris, G. Α.; Freeman, R. J. Amer. Chem. Soc., 1979, 101, 760; b) Barum, D. P.; Ernst, R. R. J. Magn. Reson., 1980, 39, 163; c) Doddrell, D. M.; Pegg, D. T. J. Amer.Chem.Soc., 1980, 102, 6390. 3. Vega, S.; Pines, A. J. Chem. Phys., 1977, 66, 5624; Wokaun, Α.; Ernst, R. R. Chem. Phys. Lett., 1977, 52, 407. 4. Bax, Α.; Freeman, R.; Frenkiel, Τ. Α.; Levitt, M. H. J. Magn. Reson., 1981, 43, 478. 5. Lauterbur, P. C. Nature, 1973, 242, 190. 6. Hoult, D. I. in "Magnetic Resonance in Biology", Ed. J.S. Cohen, Vol.1, Chapter 2, Wiley, New York 1980. 7. Damadian, R. L.; Minkov, L.; Goldsmith, M.; Koutcher, J. Naturwiss, 1978, 65, 250. RECEIVED February

1, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

2 Ultra High-Field NMR FELIX W. WEHRLI Bruker Instruments, Inc., Billerica, MA 01821 The viability of NMR spectroscopy as an analytical tool is largely owed to the on-going development of super­ conducting magnet technology which has led to magnetic fields of up to 11. than two parts in majo breakthroug combined utilization of multifilamentary NbTi and NbSn superconductors together with jointing technolo­ gies providing solenoids with very small residual resis­ tance. Paralleling these efforts were improvements in probe technology resulting in detection sensitivities that are one order of magnitude better than those achiev­ able at electromagnet fields. The resolving power of such a spectrometer permits analysis of the proton spec­ tra of very large bio-molecules while much simplifying the multiplet structure in the spectra of complex or­ ganic molecules. It is further shown that increased chemical shift dispersion greatly enhances the utility of magnetic resonance of other nuclei such as C, deuterium, and many of the quadrupolar metal resonances with inherently broad lines. 3

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Nuclear magnetic resonance spectroscopy f i r s t aroused the chemist's i n t e r e s t when the d i s c o v e r y was made that the exact nu­ c l e a r p r e c e s s i o n frequency i s dependent upon the chemical e n v i r o n ­ ment of the nucleus. The displacement of the resonance frequency r e l a t i v e to an a r b i t r a r y standard i s commonly r e f e r r e d to as chemical s h i f t . Without t h i s property, NMR would be without p r a c t i c a l u t i l i t y t o the chemist as an a n a l y t i c a l t o o l and i t would probably long be e x t i n c t . Since the chemical s h i f t i s d i c t a t e d by f i e l d - i n d u c e d para­ magnetic and diamagnetic c i r c u l a t i o n of e l e c t r o n s , i t s q u a n t i t y i s dependent upon the e x t e r n a l magnetic f i e l d o r , more a c c u r a t e l y , p r o p o r t i o n a l t o the l a t t e r . With the displacements being of the order of a few p a r t s per m i l l i o n or o f t e n only a f r a c t i o n of a p a r t per m i l l i o n , i t i s e s s e n t i a l t o conduct the experiments a t a s u f f i c i e n t l y h i g h f i e l d i n order t o r e s o l v e resonances of v a r i o u s n u c l e i p e r t a i n i n g to a molecule. 0097-6156/82/0191-0007$06.75/0 © 1982 A m e r i c a n Chemical Society

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

8

N M R SPECTROSCOPY

Another m o t i v a t i o n to i n c r e a s e the magnetic f i e l d i s detect i o n s e n s i t i v i t y . NMR happens to be an i n t r i n s i c a l l y i n s e n s i t i v e s p e c t r o s c o p i c method, being orders of magnitude l e s s s e n s i t i v e than such methods as o p t i c a l or mass spectroscopy. When NMR was f i r s t commercialized i n 1953 i n the form of a 30 MHz instrument, i t was b a r e l y p o s s i b l e to r e s o l v e the three proton resonances p e r t a i n i n g to e t h y l alcoTiol, not even to mention s p i n - s p i n c o u p l i n g . I t was t h e r e f o r e immediately recognized that i n order to become a v i a b l e technique, the r e s o l v i n g power of the instruments had to be augmented i n terms of both magnet r e s o l u t i o n and magnetic f i e l d s t r e n g t h . The e a r l y HR-30 pioneered by V a r i a n was t h e r e f o r e r a p i d l y superseded by a 40 MHz instrument. Within f i v e years the spectrometer frequency was doubled to 60 MHz, at which stage the p o p u l a t i o n of instruments r a p i d l y i n c r e a s e d , a l though the technique remained the p r i v i l e g e of few r e s e a r c h l a b o ratories. Broader use of NMR the popular A-60, an instrument that was easy to use, r e l i a b l e and which served the s c i e n t i f i c community f o r more than a decade. With the development and i n t r o d u c t i o n of the HR-100 i n 1962, NMR r e c e i v e d a f u r t h e r impetus. The r e a l breakthrough, however, was achieved by d e p a r t i n g from the c l a s s i c a l electromagnet i n 1964, although the l a t t e r succeeded i n maintaining i t s r o l e and w i l l probably not phase out b e f o r e the mid-80's. Since s a t u r a t i o n of i r o n , which i s used as the core of electromagnets, occurs between 2.0 2.3 T, other avenues had to be explored to i n c r e a s e the c r u c i a l magnetic f i e l d . The c l u e was superconducting technology, known f o r some time but not r e a l i z e d i n the form of a h i g h - r e s o l u t i o n magnet before 1964. With the i n t r o d u c t i o n of the HR-200 during that year, s h o r t l y l a t e r followed by the HR-220, a new dimension of NMR was opened up. For the f i r s t time i t became p o s s i b l e to study high-molecular-weight s y n t h e t i c and b i o l o g i c a l molecules i n s o l u t i o n , a l l o w i n g the c h a r a c t e r i z a t i o n of these complex systems by s e p a r a t i n g some of the c l o s e l y spaced resonances which cannot be r e s o l v e d at lower f i e l d s t r e n g t h . In s p i t e of t h i s remarkable achievement, superconducting spectrometers i n i t i a l l y f a i l e d to become widely popular and they r e mained the e x c l u s i v i t y of a few high-powered r e s e a r c h l a b o r a t o r i e s . There are a v a r i e t y of reasons f o r the slow s t a r t of superconducting NMR. One of the key o b s t a c l e s , which prevented e n t r y of such s y s tems i n t o the a n a l y t i c a l l a b , was the h i g h purchase p r i c e , but more importantly, the e x o r b i t a n t o p e r a t i n g c o s t s a s s o c i a t e d with the b o i l - o f f of l i q u i d helium and n i t r o g e n which, t y p i c a l l y , had to be r e p l e n i s h e d w i t h i n a few days. Moreover, these instruments r e quired s k i l l f u l o p e r a t o r s . Remarkably, however, superconducting magnets, even i n those days, were not i n f e r i o r i n r e l i a b i l i t y to t h e i r i r o n counterparts and those e a r l y systems were put out of operation p r i m a r i l y because t h e i r u t i l i z a t i o n was no longer economically viable. The n e c e s s i t y f o r improved helium economy l e d to a new genera-

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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t i o n of superconducting magnets d u r i n g the e a r l y 70's, which were f i t t e d with more e f f i c i e n t c r y o s t a t s , t h e r e f o r e p r o l o n g i n g helium and n i t r o g e n hold time. Today's c r y o s t a t s t y p i c a l l y b o i l - o f f 1020 cc of helium per hour r e s u l t i n g i n an annual helium c o s t of l e s s than $1,000 ( c f . Table I ) . Table I:

T y p i c a l annual helium consumption and a s s o c i a t e d cost f o r a superconducting magnet system between 1967 and 1980. Underlying assumption: t y p i c a l r e f i l l volume = 30 l i t e r s ; average p r i c e per l i t e r = $5.00.

YEAR

REFILL INTERVAL

ANNUAL HE COST ($)

1967 1971 1975 1980

3 Days 1 Week 3 Week

18,000 7,500 2,600

With the i n t r o d u c t i o n of Bruker's HX-270 i n 1971, f i e l d s t r e n g t h r e c e i v e d another boost. At the same time i t was recogn i z e d that h i g h magnetic f i e l d s are d e s i r a b l e and o f t e n c r i t i c a l f o r the s u c c e s s f u l o b s e r v a t i o n of a number of h e t e r o n u c l e i . Superconducting instruments t h e r e f o r e became m u l t i n u c l e a r , although they were s t i l l equipped with fixed-frequency t r a n s m i t t e r s and fixedtuned probes. Within only a few y e a r s , f i e l d s t r e n g t h was f u r t h e r increased to 300 MHz i n 1973, 360 MHz i n 1974, and f i n a l l y 400 MHz i n 1978. The l a t t e r two represent another milestone s i n c e f i e l d s beyond 300 MHz proton frequency r e q u i r e r a d i c a l l y d i f f e r e n t magnet technology. In 1979, another s i g n i f i c a n t jump was made to 500 MHz proton frequency, c o n c u r r e n t l y with the i n t r o d u c t i o n of a new generation of spectrometer consoles. T h i s culminated i n the WM-500 s p e c t r o meter (1), the h i g h e s t - f i e l d commercial NMR spectrometer b u i l t so far. I t should not go unmentioned that d u r i n g the same year r e searchers at the Carnegie-Mellon I n s t i t u t e i n P i t t s b u r g h succeeded i n t a k i n g i n t o o p e r a t i o n a 600 MHz spectrometer (2) which, however, d i f f e r s i n one important aspect; i t s f i e l d i s not p e r s i s t e n t , i . e . , the magnet has to be c o n t i n u o u s l y energized to compensate f o r f i e l d decay. The e v o l u t i o n of magnetic f i e l d s t r e n g t h i n commercial NMR spectrometers over the past 15 years i s i l l u s t r a t e d by the c h a r t i n F i g u r e 1. Magnet

Technology

In order to a p p r e c i a t e the problems behind the development of the past ten years i t may be a p p r o p r i a t e to b r i e f l y review the p r i n c i p l e s and c h a r a c t e r i s t i c s of superconductors. The m a t e r i a l s which have the magnificent property of c a r r y i n g current r e s i s t a n c e - f r e e have one element i n common: they do so only at cryogenic temperatures. Table I I l i s t s the c h a r a c t e r i s t i c s of a few commercial superconductors.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Figure L Evolution of magnetic field strength (in units of the proton magnetic resonance frequency) of commercial NMR spectrometers 1953-80. Model designations: HR (Varian), HX, WH, and WM (Bruker).

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C h a r a c t e r i s t i c s of some common superconductors

Table I I :

CRITICAL TEMPERATURE

MATERIAL NbTi Nb Sn V Ga

CRITICAL FIELD at 0°K

CRITICAL FIELD at 4,,2°K

17.6 Τ 35.0 Τ 50.0 Τ

11.8 Τ 20.0 Τ 21.1 Τ

10.6 °K 18.05°K 14.5 °K

3

3

So f a r , two p o s s i b l e routes f o r very h i g h f i e l d magnets have been pursued (_3) : (1)

Nb Sn Ribbon 3

The s t r e n g t h of t h i s approach i s i t s p r o v i s i o n of the highest c u r r e n t d e n s i t i e s weighed by a numbe (a) The diamagnetic c u r r e n t s generated a t the s u r f a c e of the conductor l e a d to an unstable o p e r a t i n g c o n d i t i o n w i t h unpre­ d i c t a b l e f i e l d / c u r r e n t r a t i o s and h i g h r e s i d u a l f i e l d s . (b) The pancake c o n s t r u c t i o n causes a discontinuous c u r r e n t path and thus higher-order f i e l d g r a d i e n t s that cannot e a s i l y be shimmed o u t . (c) J o i n i n g the tape r i n g s i s made by u s i n g soldered r e s i s ­ t i v e j o i n t s p r e c l u d i n g p e r s i s t e n t o p e r a t i o n . In p r a c t i c e t h i s means that the c u r r e n t leads cannot be removed, causing h i g h consumption of l i q u i d helium. (2)

M u l t i f i l a m e n t Nb Sn 3

Filamentary Nb Sn wire produces h i g h homogeneity and low remanence. The smaller number of j o i n t s can be made w i t h very low r e s i s t a n c e (10~~ - 10~ ohms)(3), thus p r o v i d i n g p e r s i s t e n t operation and consequently l i t t l e helium l o s s . Nb Sn filament conductors a r e g e n e r a l l y f a b r i c a t e d by means of a s o l i d - s t a t e d i f f u s i o n process ( 4 ) . In t h i s the niobium rods are p l a c e d i n t o a bronze matrix and j o i n t l y extruded. Once the f i n a l dimension i s a t t a i n e d , the wire i s subjected to heat treatment upon which t i n s e l e c t i v e l y d i f f u s e s from the c o p p e r - t i n matrix i n t o niobium. T h i s process i s o f ­ ten executed a f t e r s o l e n o i d winding s i n c e the h e a t - t r e a t e d wire can only to a l i m i t e d extent be mechanically deformed. 3

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3

The most commonly used superconductor i s NbTi, an a l l o y , which i s r e l a t i v e l y d u c t i l e , and which can be extruded i n t o wire i n a r e l a t i v e l y s t r a i g h t f o r w a r d manner. I t s t h e o r e t i c a l c r i t i c a l f i e l d at the temperature of l i q u i d helium i s 11.8 T. A more powerful superconductor i s Nb Sn, which i s superconduc3

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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t i v e below 18°K with a c r i t i c a l f i e l d of 20 Τ at the temperature of l i q u i d helium a t ambient p r e s s u r e . Another superconductor which so f a r has played only an i n s i g n i f i c a n t r o l e i n superconducting magnet technology i s V$Ga. The l a t t e r two have i n common that they are i n t e r m e t a l l i c compounds that are both extremely b r i t t l e and t h e r e f o r e most d i f f i c u l t to process* An important c r i t e r i o n of a superconductor i s i t s c r i t i c a l c u r r e n t d e n s i t y , i . e . , the l i m i t i n g value of t r a n s p o r t c u r r e n t den­ s i t y . G e n e r a l l y t h i s q u a n t i t y i s found to decrease monotonically w i t h i n c r e a s i n g f i e l d s t r e n g t h . However, even when o p e r a t i n g below the c r i t i c a l c u r r e n t , l o c a l disturbances of v a r i o u s k i n d s may p r e ­ vent a s t a b l e o p e r a t i n g c o n d i t i o n . I t i s t h e r e f o r e e s s e n t i a l that the conductor be s t a b i l i z e d . B a s i c a l l y t h i s i s achieved by embed­ ding the superconductor i n a h i g h - c o n d u c t i v i t y s u b s t r a t e m a t e r i a l ( t y p i c a l l y copper). The e f f e c t i s t h r e e f o l d : heat generated from l o c a l disturbances i s d i s s i p a t e d th heliu d th matri r i a l a c t s both as a c u r r e n penetration. A c r i t i c a requiremen superco magne p e r s i s t e n c e , i . e . , the extent of decay i n f i e l d per hour. Using s p e c i a l j o i n t i n g techniques, p e r s i s t e n c i e s of 2 p a r t s i n 10 per hour have been reached r o u t i n e l y and even values up to 1 i n 1 0 (0.05 proton Hz/hour) were a t t a i n e d ( 5 ) . If a magnet f a i l s to operate i n the p e r s i s t e n t mode, i t i s r e s i s t i v e i n e i t h e r the c o i l i t s e l f , or i n the s o - c a l l e d j o i n t s , i . e . , those c r i t i c a l i n t e r f a c e s where two wires j o i n each other. J o i n t technology, i n p a r t i c u l a r where NbaSn i s concerned, i s prob­ a b l y the most t i g h t l y kept s e c r e t of magnet manufacturers. Oxford Instruments, L t d . , which c u r r e n t l y manufactures the highest f i e l d super-homogeneous superconducting magnet o p e r a t i n g i n the p e r s i s t e n t mode, undoubtedly owes i t s l e a d to p r e c i s e l y t h i s e x p e r t i s e . The 500 MHz e t h y l benzene spectrum i n F i g u r e 2, recorded over a 30 minutes p e r i o d i n the absence of f i e l d l o c k i l l u s t r a t e s the r e ­ markable s t a b i l i t y of t h i s magnet ( 6 ) . For the c o n s t r u c t i o n of persistent-mode m u l t i f i l a m e n t magnets above 9.2 Τ a design strategy has been adopted i n which a background f i e l d of c a . 8 Τ i s generated by means of an outer s o l e n o i d c o n s i s ­ t i n g of NbTi m u l t i f i l a m e n t wire, wound on an aluminum former and potted i n a b i n d i n g matrix to prevent wire movement. I n s i d e t h i s c o i l a Nb3Sn i n s e r t c o i l i s p l a c e d that i s run i n s e r i e s with the outer s o l e n o i d . Another major challenge i n supercon magnet technology i s achievement of f i e l d u n i f o r m i t y . I t should f o r example be borne i n mind that i n order to o b t a i n a given l i n e width a t 500 MHz r e q u i r e s a 5 times b e t t e r f i e l d homogeneity than at 100 MHz spectrometer f r e ­ quency. R e a l i z a t i o n of say 0.1 Hz proton r e s o l u t i o n , a value which has been achieved and surpassed, t h e r e f o r e demands the utmost i n terms of workmanship and q u a l i t y of m a t e r i a l . T h i s concerns, among other f a c t o r s , constancy of wire diameter and u n i f o r m i t y of winding. In s p i t e of these p r e c a u t i o n s , such ambitious o b j e c t i v e s n e c e s s i t a t e v a r i o u s forms of f i e l d c o r r e c t i o n s , whose d e t a i l e d d i s c u s s i o n however, would break the scope of t h i s a r t i c l e . 8

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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PPM F/gwré? 2. /I 500-MHz proton spectrum of 0.01% ethylbenzene standard, recorded over a 30 min total accumulation time in the absence of field/frequency lock. Inset shows expanded methyl triplet (6).

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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The r e s o l u t i o n and l i n e shape t e s t s p e c t r a i n F i g u r e s 3a and b obtained a t 500 MHz demonstrate that a l e v e l o f performance has become f e a s i b l e that u n t i l r e c e n t l y was unachievable even a t a con­ s i d e r a b l y lower f i e l d . Obtainment of t h i s degree o f f i e l d u n i f o r ­ mity i s c r u c i a l , however, i f the b e n e f i t o f enhanced s h i f t separa­ t i o n i s to be f u l l y e x p l o i t e d . Instrumental Requirements a t Very High

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The magnet, though the heart of the spectrometer, i s not the only component that puts h i g h e r demands on i t s performance. A p a r t i c u l a r c h a l l e n g e , f o r example, i s probe technology, both i n terms of observation and decoupling, s i n c e a t 500 MHz one approaches the frequency range which i s midway between r a d i o frequencies and microwaves. T h i s l e d to h y b r i d c a v i t y resonators which have suc­ c e s s f u l l y been used f o r n u c l e a r broadband decoupling t a i n e d by t h i s approach. The S/N r a t i o of 20:1 on 0.01% e t h y l ben­ zene, r e c e n t l y achieved on a WM-500 spectrometer, i s n e a r l y two orders o f magnitude b e t t e r than that of a 1970 v i n t a g e electromag­ net system, thus p r o v i d i n g access to experiments on e i t h e r extremely small sample q u a n t i t i e s (submicrogram) o r a t very low concentra­ t i o n s (10~" -10~ molar). A number of a d d i t i o n a l requirements a r e r e l a t e d to the i n ­ creased s p e c t r a l windows demanding f a s t e r ADC's, l a r g e r data memo­ r i e s and d i s k storage c a p a c i t i e s and, i n order t o ensure uniform e x c i t a t i o n across the f u l l spectrum, increased pulse power. A 200 ppm F spectrum, f o r example, a t 11.7 Τ i s n e a r l y 100 KHz wide, thus r e q u i r i n g sampling a t 200 KHz. The a c h i e v a b l e r e s o l u t i o n under such circumstances i s almost c e r t a i n l y not l i m i t e d by magnet homo­ geneity but r a t h e r by data memory. From the data i n Table I I I , i t can be i n f e r r e d that a t 11.7 Τ a 250 ppm C spectrum r e q u i r e s a t l e a s t 128 Κ of data memory i n order to o b t a i n a d i g i t a l r e s o l u t i o n of .5 Hz. Although the c o s t of computer memory has s t e a d i l y de­ creased over the past y e a r s , i t may be of b e n e f i t t o enhance d i g i ­ t a l r e s o l u t i o n i n other ways than by adding memory c h i p s . T h i s can, f o r example, be r e a l i z e d by s t o r i n g the acquired data p o i n t s onto d i s k during the dwell time, i . e . , the i n t e r v a l between two d i g i t i z e r samples. T h i s s o - c a l l e d v i r t u a l memory c a p a b i l i t y allows one to a c q u i r e very l a r g e data t a b l e s and i t s only l i m i t a t i o n l i e s i n spec­ t r a l width s i n c e the maximum r a t e a t which data can be sampled and stored i s d i c t a t e d by the d i s c t r a n s f e r r a t e . For a modern h i g h ­ speed d i s c d r i v e , t r a n s f e r r a t e s are such that i n the d i s c a c q u i s i ­ t i o n mode s p e c t r a l widths o f t y p i c a l l y 50 KHz have become f e a s i b l e , which i s adequate f o r most h i g h - r e s o l u t i o n a p p l i c a t i o n s . The poten­ t i a l of t h i s approach i s e x e m p l i f i e d by the u l t r a - h i g h - r e s o l u t i o n spectrum of e t h y l benzene i n F i g u r e 4 obtained by d i s k a c q u i s i t i o n of 256 Κ data p o i n t s (6). In t h i s remarkable spectrum almost a l l of the aromatic protons are completely separated, showing l o n g range couplings between methylene and r i n g protons. 5

6

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Figure 3a.

Λ 500-MHz lineshape test showing chloroform line width at a height and at a height 1/5 thereof. Recorded on a 5-mm o.d. sample.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Figure 3b. A 500-MHz resolution test showing the high-frequency half of the o-dichlorobenzene signal. Recorded on a 5-mm o.d. sample.

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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Figure 4a. A 500-MHz proton NMR spectrum of 1% ethylbenzene obtained by in-core acquisition and transformation of 32 Κ data points without further data manipulation. Inset shows expansion of aromatic region.

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982. 7.25

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Figure 4b. Aromatic portion of the spectrum of the same sample recorded by disk acquisition and transformation of 128 Κ data points using a total of 48 Κ hardware memory. Resolution was further enhanced by Lorentz-Gauss transformation (6).

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Table I I I : Data memory requirements f o r a d i g i t a l r e s o l u t i o n of 0.5 Hz across a 250 ppm C spectrum a t d i f f e r e n t spectrometer f r e q u e n c i e s . 1 3

FIELD (T) 2.3 5.7 11.7

A

H FREQUENCY (MHz)

MEMORY CAPACITY (K WORDS) 32 64 128

100 250 500

The transformation time of such l a r g e data s e t s becomes of the order of minutes, e v e n t u a l l y i n v o k i n g hardwired F o u r i e r transform p r o c e s s o r s . Although c u r r e n t l y not i n use on any commercial NMR spectrometers, array processors are expected to supersede F o u r i e r transformation by softwar Concomitant w i t h th ments f o r permanent storage w i l l render i t necessary t o expand the storage c a p a c i t y of backup s t o r e s . D i s k storage c a p a c i t i e s of over 100 M-byte, as they have become a v a i l a b l e i n the form f o r example of the CDC 9730 S e r i e s , are a true n e c e s s i t y i n many h i g h - f i e l d NMR a p p l i c a t i o n s . Memory requirements are p a r t i c u l a r l y severe i n twodimensional spectroscopy such as the 2D-J experiments where s p e c t r a l a r r a y s are obtained i n which s p i n - s p i n c o u p l i n g i s separated from chemical s h i e l d i n g . A 10 ppm χ 50 Hz 2D spectrum w i t h a d i g i t a l r e s o l u t i o n of 2 Hz on the 6 a x i s and 0.2 Hz on the J a x i s , f o r ex­ ample, corresponds to a 4 M-word data matrix. An a d d i t i o n a l consequence of the widened s p e c t r a l windows a t high spectrometer f i e l d i s the requirement f o r enhanced pulse power. In order to assure uniform e x c i t a t i o n across the f u l l s p e c t r a l width the r f p u l s e s have to be s u f f i c i e n t l y s h o r t . T h i s i s p a r t i c u l a r l y c r i t i c a l to achieve w i t h high-Q m u l t i n u c l e a r probes, and a f u l l y s a t i s f a c t o r y s o l u t i o n to t h i s problem has a t t h i s time not been found. In F i g u r e 5 the t r a n s m i t t e r r f amplitude d i s t r i b u t i o n f o r a 25 microsecond p u l s e i s i l l u s t r a t e d f o r a 250 ppm C spectrum at three d i f f e r e n t f i e l d s t r e n g t h s , showing only minimal droop across the spectrum at 2.3 Τ but a c o n s i d e r a b l e f a l l - o f f a t 11.7 T. In p r a c t i c e , t h i s problem i s a l l e v i a t e d s i n c e r e l a x a t i o n o f t e n demands pulse f l i p angles that are c o n s i d e r a b l y l e s s than 90°. Moreover, i t turns out that the f a l l - o f f of r f amplitude i s l a r g e l y o f f s e t by the increased e f f e c t i v e r f f i e l d that e x i s t s a t f r e q u e n c i e s more remote from the c a r r i e r . 1 3

Typical Applications C l e a r l y the greatest b e n e f i t s of very h i g h magnetic f i e l d are expected i n proton spectroscopy o f l a r g e b i o l o g i c a l molecules such as p e p t i d e s , p r o t e i n s , n u c l e i c a c i d s , e t c . Organic chemists however, who have s t r u g g l e d w i t h the a n a l y s i s of proton s p e c t r a of n a t u r a l products and t h e i r s y n t h e t i c analogs w i l l agree that the s p e c t r a o f r a t h e r low-molecular weight molecules (MW 200-500) may be e n t i r e l y

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982. 13

Figure 5. amplitude distribution across a 250-ppm C NMR spectral window for a 25-ps excitation pulse at three different magnetic field strengths. Percentage numbers indicate total variation of amplitude.

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u n i n t e r p r e t a b l e unless nature has b l e s s e d them with at l e a s t one or s e v e r a l e l e c t r o n e g a t i v e s u b s t i t u e n t s or m u l t i p l e bonds. In t h i s unfortunate but r a t h e r common s i t u a t i o n two dozen m a g n e t i c a l l y nonequivalent protons may t y p i c a l l y be squeezed i n the r e g i o n between 1 and 2.5 ppm. Not only are such s p e c t r a h i g h l y second order, but worse, t h e i r i n d i v i d u a l m u l t i p l e t s are mutually overlapping. The expert NMR s p e c t r o s c o p i s t r a p i d l y concludes i n such a s i t u a t i o n that any f u r t h e r e f f o r t i s a waste of time. Figure 6 i l l u s t r a t e s the power of 500 MHz H NMR of a medium-sized molecule (7) . Among the 18 m a g n e t i c a l l y nonequivalent groups of protons i n g u a j o l (MW= 222), 16 give r i s e to separated m u l t i p l e t s i n s p i t e of t h e i r t o t a l resonance range of only 1.6 ppm. F i g u r e 6b shows the expanded spectrum f o l l o w i n g a Lorentz-Gauss transformation of the f r e e induct i o n decay, a l l o w i n g e x t r a c t i o n of three and more d i f f e r e n t s p i n s p i n c o u p l i n g constants per m u l t i p l e t . The highest f i e l d proton, f o r example, shows c o u p l i n ( v i c i n a l a x i a l - a x i a l ) an the C-4 s u b s t i t u e n t to be e q u a t o r i a l l y disposed, t h i s proton has to be assigned to e i t h e r H ( 2 ) or H(3)ax s i n c e the m u l t i p l i c i t y p a t t e r n shows two equal e q u a t o r i a l i n t e r a c t i o n s and one each of the a x i a l - a x i a l and geminal type. Since assignment of H ( l ) i s s t r a i g h t forward, a s i n g l e double resonance experiment would e l i m i n a t e t h i s ambiguity and at the same time corroborate the stereochemistry at C ( l ) or C(4). Among the many a p p l i c a t i o n s of the technique, C NMR has been found to be the method of choice f o r the c h a r a c t e r i z a t i o n of synt h e t i c polymers such as sequence a n a l y s i s , end group determination and e l u c i d a t i o n of s t e r e o r e g u l a r i t y i n v i n y l polymers. In a v i n y l polymer, f o r example, the CH s h i e l d i n g i n the -CHX-CH2- r e p e a t i n g u n i t depends upon the c o n f i g u r a t i o n of the asymmetric carbon of i t s neighbors and nearest neighbors. I f only nearest neighbors can be d i s t i n g u i s h e d , t r i a d s p l i t t i n g s are observed, according to the stereochemical d i s p o s i t i o n s mm (meso meso), mr, rm (meso racemic or i t s i n d i s t i n g u i s h a b l e racemic meso) and r r . I f next nearest i n t e r a c t i o n s can be d i f f e r e n t i a t e d , s o - c a l l e d pentad s p l i t t i n g s r e s u l t (maximum 10). Increased f i e l d c l e a r l y augments the c a p a b i l i t y of r e s o l v i n g t h i s f i n e s t r u c t u r e and the spectrum of a t a c t i c poly ( v i n y l c h l o r i d e ) i n 1,4-dioxane i n F i g u r e 7 (8) shows evidence of heptad f i n e s t r u c t u r e , i . e . , l i n e s r e s u l t i n g from c o n t r i b u t i o n s of r e p e a t i n g u n i t s i n t h i r d p o s i t i o n r e l a t i v e to the carbon observed. In t h i s case, 16 out of the t h e o r e t i c a l 36 observâtionally d i f f e r e n t heptads could be f u l l y r e s o l v e d . Another nucleus of c o n s i d e r a b l e a n a l y t i c a l p o t e n t i a l i s deuterium, whose observation at n a t u r a l abundance has been demonstrated e a r l i e r (9) but whose p r a c t i c a l i t y has so f a r been s e v e r e l y l i m i t e d due to i t s low NMR r e c e p t i v i t y and a l s o because of i t s more than s i x times smaller chemical s h i f t range r e l a t i v e to the proton. These l i m i t a t i o n s are l a r g e l y overcome at h i g h magnetic f i e l d as i l l u s t r a t e d by the natural-abundance deuterium s p e c t r a of camphor i n F i g u r e 8. By l i n i n g up the proton-decoupled deuterium spectrum with the X

a x

1 3

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982. 1

Figure 6a. A 500-MHz H full spectrum of guajol with partial assignments. Resolution in this spectrum was further enhanced by a Lorentz-Gauss transformation of the free induction decay (7).

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Figure 6b. 500-MHz *H spectrum of guajol with partial assignments. Region between 1.3 and 1.8 ppm expanded. Resolution in this spectrum was further enhanced by a Lorentz-Gauss transformation of the free induction of decay (7).

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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Figure 7. A 125.8-MHz C NMR spectrum of the methane carbon region in polyvinyl chloride, 10% in 1,4-dioxane-d at 370 K. Configurational assignments are based on the relative triad inten­ sities assuming Bernoullian statistics ($).

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Figure 8. A, A 360-MHz proton NMR spectrum of camphor, 2 M in deuterochloroform; Β and C, A 55-MHz natural-abundance H NMR spectrum of the same sample with (B), and without (C) proton broadband decoupling. 2

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

N M R SPECTROSCOPY

26

r e s p e c t i v e proton spectrum on the same ppm s c a l e (Figures 8a andb) the 1:1 correspondence of the two s p e c t r a becomes apparent. Highr e s o l u t i o n deuterium NMR s p e c t r a may t h e r e f o r e be drawn upon to e s t a b l i s h chemical s h i f t s i n complex proton s p e c t r a to provide a set of s t a r t i n g parameters f o r computer-aided s p e c t r a l a n a l y s i s . Further d i a g n o s t i c information may be derived from the protoncoupled deuterium spectrum as i l l u s t r a t e d i n F i g u r e 8c showing m u l t i p l e t s due to geminal H- H c o u p l i n g ; f o r example t r i p l e t s f o r methyl groups and doublets f o r the s i g n a l s due to the i s o l a t e d hydrogens H(2)eq and H ( 2 ) . Other m u l t i p l e t s remain unresolved s i n c e the geminal couplings are obscured by v i c i n a l and long-range c o u p l i n g . As quadrupole r e l a x a t i o n dominates deuterium r e l a x a t i o n , h i g h r e s o l u t i o n H NMR i n l i q u i d s w i l l be confined to small and mediums i z e d molecules where r e l a x a t i o n broadening i s moderate. Assuming f o r example a c o r r e l a t i o n time of 1 0 sec as i t i s t y p i c a l of a molecule of MW - 250, a based upon a quadrupole A somewhat e s o t e r i c a p p l i c a t i o n of deuterium NMR, which i s t o t a l l y outside the realm of the p o s s i b l e at low f i e l d concerns the determination of the anisotropy of the diamagnetic s u s c e p t i b i l i t y . T h i s experiment, f i r s t suggested by Dutch workers MacLean and Lohman, (10) i s based upon the f a c t that the s t a t i c quadrupole s p l i t t i n g may not be t o t a l l y averaged due to p a r t i a l alignment of the molecule at h i g h magnetic f i e l d s t r e n g t h . While d i p o l a r s p l i t t i n g s are too small to be r e t a i n e d during motional averaging, the quadrup o l a r i n t e r a c t i o n i s l a r g e enough to be d e t e c t a b l e at f i e l d s above ca. 9.2 T. F i e l d strength i s c r i t i c a l since the s p l i t t i n g s are prop o r t i o n a l to the square of the magnetic f i e l d . F i g u r e 9 shows the resolution-enhanced 77 MHz (11.7 T) deuterium spectrum of nitrobenzene d i s p l a y i n g quadrupole s p l i t t i n g s f o r a l l three magnetically nonequiv a l e n t deuterons (11). There are many more n u c l e i whose p o t e n t i a l cannot be e x p l o i t e d unless the chemical s h i f t s are d i s p e r s e d beyond the l i n e widths. T h i s holds true f o r the great d e a l of quadrupolar n u c l e i which cons t i t u t e the bulk of the magnetic n u c l e i i n the P e r i o d i c Table and which play an important r o l e i n i n o r g a n i c chemistry. The u t i l i t y of h i g h magnetic f i e l d i n t h i s area may be i l l u s t r a t e d with an example from i n o r g a n i c s o l u t i o n chemistry, r e l a t e d to some recent work i n t h i s l a b o r a t o r y d i r e c t e d toward the charact e r i z a t i o n of the s o l u t i o n species formed upon d i s s o l v i n g the a l u minum h a l i d e s i n p o l a r organic s o l v e n t s (12). While, f o r a long time, i t was assumed that i n p o l a r s o l v e n t s the dimeric A I 2 C I 6 breaks up i n t o A l C l i f " and A 1 S 6 (S = a c e t o n i t r i l e ) , t h i s view was r e c e n t l y challenged (13) as a r e s u l t of the A 1 s p e c t r a which suggest the e x i s t e n c e of mixed species [ A l C l S 6 _ ] 3 ~ . However, c o n c l u s i v e evidence was only provided by experiments at high f i e l d . F i g u r e 10 shows a stacked p l o t of 93.7 MHz A 1 NMR s p e c t r a of A I C I 3 i n a c e t o n i t r i l e recorded at d i f f e r e n t solute concentrations. These s i g n a l s are due to above-mentioned hexacoordinated s o l v a t e / c o u n t e r i o n complexes which could be i d e n t i f i e d on the b a s i s of c h a r a c t e r i s t i c chemical s h i f t s and p r e d i c t a b l e 2

X

a x

2

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27

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27

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Figure 9. A 76.8-MHz resolution-enhanced deuterium NMR spectrum of neat nitrobenzene showing quadrupole splittings due to partial alignment resulting from anisotropy of diamagnetic susceptibility. (Reproduced, with permission, from Ref. 11. Copyright 1981, Academic Press.)

to

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Figure 10. High-field Al NMR spectra of aluminum chloride in acetonitrile solu­ tion showing the resonance region of hexacoordinated Al. Peak designations: 1, Al(CH CN) ; 2, [AlCl(CH CN) ] +; 3, c\sr[AlCl (CH CN)J*; 4, tranS'[AlClg(CH CN)t] ; 5, cis-AlCl (CH CN) ; 6, trans-AlCl (CH CN) , and 7, cis/trans[AlCl (CH CN)zY. (Reproduced with permission, from Ref. 12. Copyright 1981, Academic Press.) 27

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

2.

WEHRLI

Ultra

High-Field

NMR

29

quadrupole r e l a x a t i o n r a t e s . The c l o s e l y spaced l i n e p a i r s 3/4 and 5/6, f o r example, r e f l e c t the e f f e c t of c i s / t r a n s isomerism i n [AICI2S1J" " and A I C I 3 S 3 which would c l e a r l y be u n r e s o l v a b l e i f the f i e l d s t r e n g t h were halved. In c o n c l u s i o n , i t may be stated that NMR a t very h i g h f i e l d has given the technique an unprecedented impetus. As a consequence of the enhanced s h i f t s e p a r a t i o n the s p e c t r a l i n f o r m a t i o n content has been g r e a t l y increased, opening up a range of new experiments. Together w i t h the concomitant s e n s i t i v i t y increase t h i s w i l l have a s i g n i f i c a n t impact on a v a r i e t y of r e s e a r c h areas, notably molecular b i o l o g y , macromolecular chemistry, but a l s o organic and i n o r g a n i c chemistry i n g e n e r a l . 1

LITERATURE CITED 1. Bruker Report 3/1979 2. 600 MHz Symposium Carnegie-Mello , 3. McDonald, P. & Proctor, W., Proceedings of the Eighth Inter­ national Cryogenic Engineering Conference, 1980, p. 509. 4. Kwasnitza, Κ., Narlikar, A.V., Nissen, H.U., & Salate, D., Cryogenics 20, 715, 1980. 5. Hull, W. E., personal communication. 6. Hull, W. E., personal communication. 7. Formacek, V., personal communication. 8. Elgert, K.F., Kosfeld, R., & Hull, W.E., personal communication. 9. Shoolery, J.N., Varian Application Topic, 1977, p. 7-14. 10. Lohman, J.A.B., & MacLean, C., Chem. Phys., 1978, 35, p. 269; 1979, 43, 144. 11. Lohman, J.A.B., MacLean, C., J. Magn. Res., 1981, 42, p.5. 12. Wehrli, F.W. & Wehrli, S.L., J. Magn. Res., 1981, 44. 13. Akitt, J.W. & Duncan, R.H., J. Magn. Res., 1977, 25, 391. RECEIVED February 2, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

3 N M R Spectroscopy at 600 M H z A. A. BOTHNER-BY and J. DADOK Carnegie-Melon University, Department of Chemistry, Pittsburgh, PA 15213

In December 1979 th NMR Facilit fo Biomedical Studie formally opened servic there, and the intervening year has seen a variety of applications to structure made possible by the high magnetic field (14.1 Tesla) employed. The first advantage which comes to mind when considering the use of high magnetic fields for NMR is the increased spectral dispersion which results, and the possibility of separating and observing signals from complex molecules which are not resolved at lower fields. This advantage is well understood and widely appreciated, and only a few illustrative examples will be given here. An example of the degree of complexity which can be sorted out is Palytoxin, a water-soluble toxin obtained from marine coelenterates of the genus Palythoa. The molecular formula of the compound is C H N O . The complete gross structure for Palytoxin has been elucidated by R. E. Moore and G. Bartolini of the University of Hawaii and reported in a recent communication (1). The structural elucidation involved extensive proton spectroscopy at 600 MHz, together with homonuclear decoupling, to establish the sequence and stereochemistry in degradation frag­ ments as well as the entire molecule. The structure deduced is displayed in Figure 1, with the spectrum of the intact toxin shown beneath. An expanded view of a portion of this spectrum is displayed in Figure 2. The structure of an N-terminal degradation product and its spectrum is displayed in Figure 3. A second example illustrating the greater dispersion at high field is bradykinin, a nonapeptide with sequence arg-pro-pro-glyphe-ser-pro-phe-arg. The spectrum of this peptide and of a number of analogs has been investigated in a collaborative effort with Dr. G. Fisher of the University of Miami. The spectrum with assignments is shown in Figure k. The separate observation of β protons from each of the three proline residues has made it possible, for example, to determine the degree of puckering and preference for Ramachandran A or Β forms in proline rings. Analysis of coupling constants in the side chains, amide proton 129

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0 0 9 7 - 6 1 5 6 / 8 2 / 0 1 9 1 -0031 $ 0 6 . 0 0 / 0 © 1982 A m e r i c a n C h e m i c a l Society

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

NMR

32

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Figure 1. A 600-MHz high resolution spectrum of palytoxin, a marine toxin isolated from a species of Palythoa. The gross structure deduced for the toxin also is shown.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

3.

B O T H N E R - B Y A N D DADOK

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33

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Expanded view of a small region of the spectrum of palytoxin shown in Figure 1.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Figure 3.

Structure of terminal fragment of palytoxin obtained from the N-p-bromobenzoyl rivative via periodate cleavage and spectrum thereof.

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A 600-MHz spectrum of bradykinin in D O solution. z

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

900

N M R SPECTROSCOPY

36

s h i f t s , pH t i t r a t i o n , use o f paramagnetic s h i f t and broadening reagents, and other standard NMR techniques have been a p p l i e d to demonstrate that b r a d y k i n i n i s a r a p i d l y i n t e r c o n v e r t i n g mixture of many conformers, w i t h no h i g h l y p r e f e r r e d conformation i n s o l u t i o n , but that complexes are formed w i t h l i p i d s , p a r t i c u l a r l y at pro^ and phe° which impose some conformational r e s t r a i n t on the molecule ( 2 ) . Other c l a s s e s o f compounds f o r which the e x t r a r e s o l v i n g power at h i g h f i e l d has been u s e f u l i n c l u d e DNA r e s t r i c t i o n f r a g ­ ments ( i n v e s t i g a t e d by T. A. E a r l y , D. R. Kearns, W. H i l l e r and R. D. Wells (2); a l k a l o i d s (S. Danishefsky (kj), and sugars (£). A second advantage o f h i g h e r f i e l d s i s that r a p i d chemical exchange processes ( f o r example between conformational s t a t e s ) are more r e a d i l y apparent. L i n e broadening i n the r a p i d exchange l i m i t i s given by: Δν where increased l i n e width, P A and p are the f r a c ­ t i o n s o f the two conformers, (v^-Vg) the chemical s h i f t i n frequency u n i t s between n u c l e i i n the two conformers, and τ the average r e s i d e n c e time i n one conformer. Increasing the f i e l d by a f a c t o r of t e n w i l l increase the l i n e broadening by a f a c t o r of one hundred; r a p i d exchange processes which cause a broadening o f 0 . 1 Hz at 60 MHz and might escape n o t i c e , w i l l cause a broadening of 10 Hz at 600 MHz and w i l l be very n o t i c e a b l e . Thus P r o f e s s o r J . G l a s e l ( 6 ) noted that the 600 MHz spectrum of morphine at p h y s i o l o g i c a l pH was broad ( F i g u r e 5 ) , (although the low f i e l d spectrum appeared normal). This suggested a con­ formational e q u i l i b r i u m (axial-N-methyl Ζ equatorial -N-methyl). The c o n c l u s i o n was confirmed by observing the spectrum at pH 1. 5 , which slowed the i n t e r c o n v e r s i o n and gave a sharp spectrum show­ ing s i g n a l s from each conformer. The i n t e n s i t i e s were i n the r a t i o 1 : 5 , i n d i c a t i n g an energy d i f f e r e n c e of o n l y about 1 K c a l / mole, although e a r l i e r c a l c u l a t i o n s (7) had p r e d i c t e d the Ne q u a t o r i a l conformer to be more s t a b l e by about 6 Kcal/mole. The i n t e r e s t i n g question a r i s e s as to which conformer i s bound i n the r e c e p t o r s i t e , and the r o l e o f the lone p a i r i n binding. The a v a i l a b i l i t y of h i g h e r f i e l d s a l s o opens the door to the o b s e r v a t i o n o f features i n the NMR spectrum which depend quadrati c a l l y on the f i e l d strength. In p a r t i c u l a r , the f i e l d i s s u f f i c i e n t l y strong so that even r e l a t i v e l y small diamagnetic and paramagnetic molecules w i t h a n i s o t r o p i c magnetic s u s c e p t i b i l i t i e s are s i g n i f i c a n t l y o r i e n t e d , and the o r d e r i n g produces observable e f f e c t s i n the spectrum. Such e f f e c t s have been observed i n the past w i t h molecules i n macroscopic c r y s t a l s which are e i t h e r p h y s i c a l l y o r i e n t e d , or are o r i e n t e d by the f i e l d , ( 8 ) or by observing the s p e c t r a of the molecules d i s s o l v e d i n l i q u i d cry­ s t a l s ( i n which the l i q u i d c r y s t a l domains are o r i e n t e d by the f i e l d ) , (2) o r by the a p p l i c a t i o n o f a strong e l e c t r i c f i e l d t o s o l u t i o n s of p o l a r molecules ( 1 0 ) . i s

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In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

NMR Spectroscopy at 600

B O T H N E R - B Y A N D DADOK

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Figure 5. Spectra of morphine at neutral and low pH demonstrating the occur­ rence of exchange phenomena at neutral pH (a) and slow inter conversion of axial and equatorial N-methyl isomers at low pH (b).

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

38

NMR

SPECTROSCOPY

F o l l o w i n g the treatment o f Lohman and MacLean, (jLl) the o r d e r i n g of the molecule may be described i n terms of two order­ ing parameters S and K, which are defined by the r e l a t i o n s S = 2

2 Κ = ( s i n θ cos 2 φ ) The angular brackets denote an e x p e c t a t i o n or time-averaged value, and θ and φ are the p o l a r angles g i v i n g the d i r e c t i o n of the s t a t i c magnetic f i e l d i n the molecule-fixed coordinate frame x,y,z. These axes are chosen to be the p r i n c i p a l axes of the magnetic s u s c e p t i b i l i t y tensor o f the molecule. The energy o f the molecule i n a magnetic f i e l d i s given by the expression

The o r d e r i n g parameters S and Κ may be c a l c u l a t e d assuming a Boltzmann d i s t r i b u t i o n , and are then given by S = ΔχΗ^/^Τ

Κ = ALx«P/l5kT where Δχ the s u s c e p t i b i l i t y anisotropy i s given by χ -l/2 (Xxx Xyy)> Χ s u s c e p t i b i l i t y asymmetry i s given by Xxx " Xyy' po ^. For aromatic molecules, Δχ amounts to about 1 χ 1 0 " ^ ° cnP/mol per benzene r i n g ( 1 2 ) , w h i l e Δ χ i s expected to be an order of magnitude smaller, so the o r d e r i n g parameter S f o r benzene at room temperature and lkl k i l o g a u s s i s expected to be about 3 χ ΙΟ- . ζ ζ

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S u b s t i t u t i n g t y p i c a l values of Δ μ , the parameter i s found to be only one to two orders o f magnitude l a r g e r than those of aromatic compounds. The occurrence o f t h i s o r d e r i n g can be detected i n s e v e r a l ways u s i n g magnetic techniques. In the case of quadrupolar n u c l e i , such as deuterium, the o r d e r i n g w i l l give r i s e to a quadrupole s p l i t t i n g of the deuteron s i g n a l , as i s w e l l known from experiments with l i q u i d c r y s t a l s (Γ5). The o b s e r v a t i o n of l a r g e s p l i t t i n g s i n a small paramagnetic molecule a r i s i n g from t h i s o r i g i n have been reported by P. J . Domaille (1*0, example showing such s p l i t t i n g s i s given i n Figure"^. The o b s e r v a t i o n of deuteron quadrupole s p l i t t i n g s i n the deuteron s p e c t r a of deuterated aromatic compounds has a l s o been reported i n a s e r i e s o f a r t i c l e s by Lohman and Maclean ( l l , l j ? , l 6 ) . a n d

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

a

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NMR Spectroscopy at 600

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