Lecture #7 (2D NMR) Utility of Resonance Assignments

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "Lecture #7 (2D NMR) Utility of Resonance Assignments"

Transcription

1 Lecture #7 (2D NMR) Basics of multidimensional NMR (2D NMR) 2D NOESY, COSY and TOCSY 2/23/15 Utility of Resonance Assignments Resonance Assignments: Assignment of frequency positions of resonances (peaks) in the NMR spectrum to specific atoms in the macromolecule. Macromolecule H Protein folding Resonance Assignments Dynamics (10-12 to seconds time scale) 1D 1H NMR spectrum Protein pka measurements Mapping ligand binding surfaces (drug discovery) Multidimensional experiments required to resolve peak overlap Structure 1

2 COSY: COrrelation SpectroscopY 2

3 Basic 2D COSY COSY: COrrelation SpectroscopY Review: the effect of 90 o pulses applied on different axes Before After o 90 o x pulse (B1) B 1 Bo Bo Mo Mo z x Detected component The amount of component in transverse plane is Mo sin (360* *t1) (Blue). Only this component is detected. Similar to 1D it will rotate in the transverse (xy) plane at a frequency 3

4 Peak Peak height proportional to: Mo sin (360 t 1 ) z F2 frequency domain x t 1 = 0 sec interferogram intensity Magnitude of peak maxima oscillates at sin (360* a*t 1 ) 4

5 Amplitude of peak is modulated by Cos (2 * a *t 1 ) Interferogram (the 2 nd ) a (direct dimension) a a 5

6 Case II (transfer during mixing period) t1 (F1) t2 (F2) Key experimental parameters for multidimensional NMR experiments (1) Sweep-width in indirect dimension: Controlled by the amount the t1 delay is incremented between successive experiments. (t 1 ): Amount that the t1 delay is incremented between successive experiments. SW(F1) = 1/( (t 1 )) ***Note: it s more complicated than this!! It depends upon how the experiment was performed. There are many ways to obtain quadrature detection in the indirect dimension (different ways to sample the signal so as to discern positive and negative frequencies in the indirect dimension). In these different methods (STATES, STATES-TPPI, TPPI, ECHO-ANTIECHO) the delay is not always incremented on each successive t1 increment! (t 1 )= 100 s t 1 =0 s Example: (t1)= 100 s 1 / (100 s) = 10,000Hz t 1 =100 s t 1 =200 s t 1 =300 s SW(F1) = 10,000Hz F2 (direct dimension) F1 (indirect dimension) t 1 =400 s (t 1 )= 50 s t 1 =0 s Example: (t1)= 50 s 1 / (50 s) = 20,000Hz t 1 =50 s t 1 =100 s t 1 =150 s SW(F1) = 20,000Hz F1 (indirect dimension) t 1 =200 s F2 (direct dimension) 6

7 (2) Digital resolution (DR) in indirect dimension Interferogram The more t 1 increments you collect the better the digital resolution in the indirect dimension. interferogram (t 1 ) 128 t1 increments 512 t1 increments Nyquist theorem: To properly determine the frequency of an oscillating signal must sample it at least twice per wavelength Typically 256 to 1024 experiments (increments) are recorded depending on the desired digital resolution in indirect dimension 7

8 (2) Digital resolution (DR) in indirect dimension A Indirect dimension (F2) Indirect dimension (F2) B Example: SW(F1) = 10,000 Hz expts. recorded = 128 DR= Hz/point 128 1D files (serial files) collected in t1 dimension Example: SW(F1) = 10,000 Hz expts. recorded = 512 DR = 39.1 Hz/point 512 1D files (serial files) collected in t1 dimension Mid-80s Early 90s late 90s - to present Different strategies to assign proteins Homonuclear Methods (1H 2D COSY, TOCSY etc.) Proteins < kd (< ~100 aa) Old School Triple Resonance (3D/4D 1H, 13C & 15N) (most common) Quadruple Resonance (3D/4D 1H, 13C & 15N) Proteins < 30 kd (< 250 aa) Proteins < 85 kd (< 723aa) JACS (2002) 124: Direct dimension (F2) 8

9 Important 2D experiments used in homonuclear assignment methods 2D COSY 2D NOESY 2D COSY 2D TOCSY NOESY (Nuclear Overhauser Enhancement SpectroscopY): Connects hydrogen atom that are separated by < 5 Angstroms (<0.5nanometers) TOCSY (TOtal Correlation SpectroscopY): Connects hydrogen atoms that are part of the same spin network (spin system). Some people refer to this as a HOHAHA experiment. COSY (COrrelation SpectroscopY): Connects hydrogen atoms that are separated by three bonds or less *Homonuclear: Frequency of single atom type (hydrogen in these experiments) is observed in frequency dimensions In the COSY experiment, magnetization is transferred by scalar coupling. Protons that are more than three chemical bonds apart give no cross signal because the 4 J coupling constants are close to 0. Therefore, only signals of protons which are two or three bonds apart are visible in a COSY spectrum (red signals). The cross signals between HN and Halpha protons are of special importance because the phi torsion angle of the protein backbone can be derived from the 3 J coupling constant between them. 9

10 2D TOCSY 2D NOESY In the TOCSY experiment, magnetization is dispersed over a complete spin-system (set of hydrogen atoms) of an amino acid by successive scalar coupling. The TOCSY experiment correlates all protons of a spin system. Therefore, not only the red signals are visible (which also appear in a COSY spectrum) but also additional signals (green) which originate from the interaction of all protons of a spin system that are not directly connected via three chemical bonds. In the NOESY experiment, magnetization is transferred between 1H nuclei when they are < ~5A apart The closer the nuclei the stronger the cross-peak (proportional r -6 ) 10

11 Example 2D NOESY spectrum 1 mm Protein dissolved in water (55.5M H2O) 2D NOESY Experiment (transfer of magnetization via dipole-dipole interactions) H I 1D spectrum <6 angstroms H S < 5A Ha Hb (f) (g) < 5A Hc (a) (b) (c) (d) (e) I S 0 Hz frequency Example of single increment in the experiment (a) IS (b) (c) I cos (360* S *t 1 ) S I S Hb Hc Ha F2 F1 F2 (ppm) F1 (ppm) (d) I S m period (e) I S Non-equilibrium condition. Populations of S-spin inverted Transient NOE condition. Spin S has been inverted, while spin I has not. Hc Hb F2 Ha F1 NOE effect causes intensity of I to be reduced (negative NOE). The amount of S on z axis depends on cos (360* S *t 1 ). ( its projection onto the Y axis at step (c)) Therefore the intensity reduction in I is modulated by cos (360* S *t 1 ). Wo Assuming Negative NOE (Wo dominates) 11

12 2D NOESY Experiment (transfer of magnetization via dipole-dipole interactions) (f) H I 1D spectrum <6 angstroms H S Intensity of cross-peak related to mixing time (t m ) (a) (b) (c) (d) (e) I S 0 Hz frequency (d) (f) I S m period (e) I S Intensity of I modulated by amount of S magnetization at beginning of mixing time that is inverted. This in turn depends upon larmor frequency of spin S in t1 dimension. Therefore frequency of S nucleus in F1 dimension has been encoded within the amplitude of spin I when it is detected Rate of build-up of cross-peak is inversely proportional to distance separation S Direct detection of I oscillating at frequency I I Rate of NOE build-up (t2 -> F2) (t1 -> F1) I F2 (direct dimension) S F1 (indirect dimension) ms Typical Mixing time We record NOESY spectra at a fixed tm value where intensity is proportional in inter-hydrogen atom separation (r IS ) 12

13 The sign of the cross-peak in NOESY depends on the molecular tumbling time (size) of the molecule or whether it is caused by chemical exchange. Large molecule (- NOE) Biomolecules! Wo dominates I F2 (direct dimension) S F1 (indirect dimension) Small molecule ( NOE) - I F2 (direct dimension) - H I S F1 (indirect dimension) r IS H S Cross-peaks from NOEs Protein requirements for homonuclear resonance assignment methods (1) Protein molecular weight < kds (<~100aa) homonuclear assignment methods don t work for larger proteins because: (a) Increased spectral complexity. (signal overlap) (b) Increased proton transverse relaxation (smaller T 2 values cause proton magnetization to decay too rapidly. Important homonuclear proton COSY and TOCSY experiments no longer work ) (2) protein concentration > 0.25 to 0.5mM. Data collection for less concentrated proteins takes too long. S/N is proportional to Sqrt (# of scans). 16 times as many scans need to be acquired on a 0.125mM sample to get the same S/N ratio as obtained when the same experiment is acquired on the same sample concentrated to 0.5mM. (3) Salt (NaCl or KCl) concentrations <500mM Higher salt concentrations degrade the performance of the NMR probe reducing signal to noise. (4) Must not significantly aggregate. Aggregation increases apparent c. This decreases the T2 values. Cross-peaks from chemical exchange a Chemical exchange: During the mixing time ( m) the magnetic environment of hydrogen atom changes (chemical shift changes) as a result of conformational rearrangement or chemical reaction b F1 (indirect dimension) e.g. a b Conformational change (5) dissolved in non-protonated solvents. Protonated solvents give rise to signals in the NMR spectra that mask those of the protein. Note: Most experiments are performed on samples containing water which gives rise to a water peak at ~4.76ppm. This huge signal is normally eliminated by using water suppression techniques during the experiment (e.g. selective excitation, solvent presaturation, magnetic field gradients) Some good NMR buffers: (a) 50mM PO 4 /100mM NaCl/7% 2 H 2 O (D 2 O) (b) 50mM Tris-d11/100mM NaCl/7% 2 H 2 O (D 2 O) (c) deuterated acetate/100mm NaCl/7% 2 H 2 O (D 2 O) Frequently 0.01% Sodium Azide is also added to prevent microbial growth F2 (di t di i ) 13

14 Why do we need 2 H 2 O in the solvent? The magnetic field generated by the superconducting magnet changes with time ( drifts ) 2 H 2 O is used to lock the magnetic field (keep it from changing) during data acquisition On a 500 MHz NMR magnet 1 H frequency = 500 MHz 2 H frequency = 76.8 MHz (6) Sample should be soluble at ph values < 8.0 At alkaline ph values have increased rate of exchange of protein hydrogen atoms with solvent. This tends to attenuate the signals arising from exchangeable atoms. Solvent Exchange H A N H B O H C k intr H B N H A O acid or base catalyzed H C H B H A N H B (-) (-) O O N H C H A H C Example of base catalyzed NMR Experiment Lock System H A N Observed chem. shift of H A ~ chem. shift of H 2 O H 2 O 1H RF excitation & detection (500 MHz) 2 H RF excitation & detection (76.8MHz) Computer (signal processing and storage) Automatically adjust magnetic field to counter-act field drift Monitor 2 H Signal position Chemical shift of atom H A = P H A N Fraction of N-H A ~10-3 M A P O H A H H 2 O B ~ H 2 O Fraction of H A -O-H B ~55 M 14

15 Protons that exchange H N Backbone amide Trp indole His sidechain Sample manipulation trick: Change buffer into D 2 O solvent to simplify the NMR spectra H H O H H O H H O O H Ser/Thr/Tyr Arginine sidechain N-term residue, Lys sidechain O C.. S H.. HO Carboxylic acid (C-term residue, Asp, Glu) Asn, Gln sidechain Cys sidechain N-term.. N C C N C C N C C.. CH CH HO CH 3 H 3 C CH 3 Buffer exchange protein into 100% D 2 O solvent (cycle to higher ph to facilitate exchange: ph <7 ph 8 ph <7 ) H C-term water sample 50mM PO 4 100mM NaCl 7% 2 H 2 O (D 2 O) 0.01% NaN 3 Not observable in NMR spectrum D H O D H O D H O N-term.. N C C N C C N C C.. C-term CH CH DO CH 3 H 3 C CH 3 H D2O sample 50mM PO 4 100mM NaCl 99.99% 2 H 2 O (D 2 O) 0.01% NaN 3 15

Structure Determination by NMR

Structure Determination by NMR Structure Determination by NMR * Introduction to NMR * 2D NMR, resonance assignments J Correlated Based Experiments * COSY - Correlated Spectroscopy * NOESY - Nuclear Overhauser Effect Spectroscopy * HETCOR

More information

Basic Pulse Sequences I

Basic Pulse Sequences I Basic Pulse Sequences I Pascale Legault Département de Biochimie Université de Montréal 1 Outline 1) Introduction to Multidimensional NMR 2) The 2D HSQC Experiment: Limitation of Classical Approaches 3)

More information

NMR Nuclear Magnetic Resonance

NMR Nuclear Magnetic Resonance NMR Nuclear Magnetic Resonance Nuclear magnetic resonance (NMR) is an effect whereby magnetic nuclei in a magnetic field absorb and re-emit electromagnetic (EM) energy. This energy is at a specific resonance

More information

NMR Spectroscopy in Notre Dame

NMR Spectroscopy in Notre Dame NMR Spectroscopy in Notre Dame University of Notre Dame College of Science Department of Chemistry and Biochemistry Nuclear Magnetic Resonance Facility http://www.nd.edu/~nmr Reservation system for spectrometers

More information

General Principles of Two Dimensional (2D) NMR

General Principles of Two Dimensional (2D) NMR General Principles of Two Dimensional (2D) NMR In NMR the most useful information comes from the interactions between two nuclei, either through the bonds which connect them (J-coupling interaction) or

More information

There are certain things that we have to take into account before and after we take an FID (or the spectrum, the FID is not that useful after all).

There are certain things that we have to take into account before and after we take an FID (or the spectrum, the FID is not that useful after all). Data acquisition There are certain things that we have to take into account before and after we take an FID (or the spectrum, the FID is not that useful after all). Some deal with the detection system.

More information

Heteronuclear correlation - HETCOR

Heteronuclear correlation - HETCOR Heteronuclear correlation - HETCOR Last time we saw how the second dimension comes to be, and we analyzed how the COSY experiment (homonuclear correlation) works. In a similar fashion we can perform a

More information

1 H NMR Spectra of Proteins

1 H NMR Spectra of Proteins NMR of Proteins Determining Protein Structures by NMR the process of determining a solution structure by NMR is one of measuring many (hundreds/thousands) of short protonproton distances and angles, and

More information

2D NMR: Applications. IUPAB sponsored Workshop on NMR & its Applications in Biological Systems November 23-30, Mamata Joshi TIFR, Mumbai

2D NMR: Applications. IUPAB sponsored Workshop on NMR & its Applications in Biological Systems November 23-30, Mamata Joshi TIFR, Mumbai 2D NMR: Applications IUPAB sponsored Workshop on NMR & its Applications in Biological Systems November 23-30, 2009 Mamata Joshi TIFR, Mumbai What is 2D NMR? When and Why? How to apply? Stack of several

More information

Experiments for Backbone and Sidechain Assignments of Uniformly 13 C- and 15 N- Labeled Proteins

Experiments for Backbone and Sidechain Assignments of Uniformly 13 C- and 15 N- Labeled Proteins Experiments for Backbone and Sidechain Assignments of Uniformly 13 C- and 15 N- Labeled Proteins Class Outline Understanding 3D experiments Chemical shift assignment experiments Backbone Aliphatic sidechains

More information

NMR is the most powerful structure determination tool available to organic chemists.

NMR is the most powerful structure determination tool available to organic chemists. Nuclear Magnetic esonance (NM) Spectrometry NM is the most powerful structure determination tool available to organic chemists. An NM spectrum provides information about: 1. The number of atoms of a given

More information

Assume MAS on powders for all problems, unless stated otherwise (or obvious from the context). Calculation Exercise #1 (Wednesday)

Assume MAS on powders for all problems, unless stated otherwise (or obvious from the context). Calculation Exercise #1 (Wednesday) Exercises for Solid-State NMR Spectroscopy in Materials Chemistry Mattias Edén, Department of Materials and Environmental Chemistry, Stockholm University Assume MAS on powders for all problems, unless

More information

Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance Spectroscopy Nuclear Magnetic Resonance Spectroscopy Introduction NMR is the most powerful tool available for organic structure determination. It is used to study a wide variety of nuclei: 1 H 13 C 15 N 19 F 31 P 2

More information

Signal Manipulation. time domain NMR signal in MHz range is converted to khz (audio) range by mixing with the reference ( carrier ) frequency

Signal Manipulation. time domain NMR signal in MHz range is converted to khz (audio) range by mixing with the reference ( carrier ) frequency NMR Spectroscopy: 3 Signal Manipulation time domain NMR signal in MHz range is converted to khz (audio) range by mixing with the reference ( carrier ) frequency Ref in (MHz) mixer Signal in (MHz) Signal

More information

NMR for Physical and Biological Scientists Thomas C. Pochapsky and Susan Sondej Pochapsky Table of Contents

NMR for Physical and Biological Scientists Thomas C. Pochapsky and Susan Sondej Pochapsky Table of Contents Preface Symbols and fundamental constants 1. What is spectroscopy? A semiclassical description of spectroscopy Damped harmonics Quantum oscillators The spectroscopic experiment Ensembles and coherence

More information

Proton Nuclear Magnetic Resonance Spectroscopy

Proton Nuclear Magnetic Resonance Spectroscopy CHEM 334L Organic Chemistry Laboratory Revision 2.0 Proton Nuclear Magnetic Resonance Spectroscopy In this laboratory exercise we will learn how to use the Chemistry Department's Nuclear Magnetic Resonance

More information

VTT TECHNICAL RESEARCH CENTRE OF FINLAND

VTT TECHNICAL RESEARCH CENTRE OF FINLAND Figure from: http://www.embl.de/nmr/sattler/teaching Why NMR (instead of X ray crystallography) a great number of macromolecules won't crystallize) natural environmant (water) ligand binding and inter

More information

Two-Dimensional Nuclear Magnetic Resonance: Principle and Applications. Guangjin Hou

Two-Dimensional Nuclear Magnetic Resonance: Principle and Applications. Guangjin Hou Two-Dimensional Nuclear Magnetic Resonance: Principle and Applications Guangjin Hou 0.07.008 Outlines Histories of Multi-Dimensional NMR The general scheme for D NMR The classes of D NMR experiments -D

More information

Chemistry 24b Lecture 21&22 Spring Quarter 2004 Instructor: Richard Roberts

Chemistry 24b Lecture 21&22 Spring Quarter 2004 Instructor: Richard Roberts Chemistry 4b Lecture & Spring Quarter 004 Instructor: Richard Roberts Two Dimensional NMR The experiments and spectra so far described are one dimensional in the sense that the FID obtained is a function

More information

13C NMR Spectroscopy

13C NMR Spectroscopy 13 C NMR Spectroscopy Introduction Nuclear magnetic resonance spectroscopy (NMR) is the most powerful tool available for structural determination. A nucleus with an odd number of protons, an odd number

More information

Introduction to 1D and 2D NMR Spectroscopy (1) Basics

Introduction to 1D and 2D NMR Spectroscopy (1) Basics Introduction to 1D and 2D NMR Spectroscopy (1) Basics Lecturer: Weiguo Hu A328 Conte (7-1428) weiguoh@polysci.umass.edu October 2009 1 Content At a Glance Introduction to 1D and 2D NMR Spectroscopy Experimentation

More information

Tetramethylsilane (TMS) Trimethylsilyl d 4. -propionic acid (TMSP) Dioxane. O - Na + Dimethylfura n. Potassium Hydrogen Phthalate. Sodium Maleate CH 3

Tetramethylsilane (TMS) Trimethylsilyl d 4. -propionic acid (TMSP) Dioxane. O - Na + Dimethylfura n. Potassium Hydrogen Phthalate. Sodium Maleate CH 3 Practical Aspects of Quantitative NMR Experiments This discussion presumes that you already have an understanding of the basic theory of NMR. There are a number of issues that should be considered when

More information

C NMR Spectroscopy C NMR. C Transition Energy

C NMR Spectroscopy C NMR. C Transition Energy NMR NMR Spectroscopy is the most abundant natural isotope of carbon, but has a nuclear spin I = 0, rendering it unobservable by NMR. Limited to the observation of the nucleus which constitutes only.% of

More information

Generation and Detection of NMR Signals

Generation and Detection of NMR Signals Generation and Detection of NMR Signals Hanudatta S. Atreya NMR Research Centre Indian Institute of Science NMR Spectroscopy Spin (I)=1/2h B 0 Energy 0 = B 0 Classical picture (B 0 ) Quantum Mechanical

More information

Organic Chemistry Tenth Edition

Organic Chemistry Tenth Edition Organic Chemistry Tenth Edition T. W. Graham Solomons Craig B. Fryhle Welcome to CHM 22 Organic Chemisty II Chapters 2 (IR), 9, 3-20. Chapter 2 and Chapter 9 Spectroscopy (interaction of molecule with

More information

Basic principles of multidimensional NMR in solution

Basic principles of multidimensional NMR in solution Basic principles of multidimensional NMR in solution 30.09.2009 The program 2/78 General aspects Basic principles Parameters in NMR spectroscopy Multidimensional NMR-spectroscopy Protein structures NMR-spectra

More information

Introduction to Nuclear Magnetic Resonance (NMR) And. NMR Metabolomics

Introduction to Nuclear Magnetic Resonance (NMR) And. NMR Metabolomics Introduction to Nuclear Magnetic Resonance (NMR) And NMR Metabolomics Acknowledgment: Some slides from talks by Natalia Serkova, Wimal Pathmasiri, and from many internet sources (e.g., U of Oxford, Florida

More information

Christ Church 3 rd Year: Magnetic Resonance. Reading. Topics

Christ Church 3 rd Year: Magnetic Resonance. Reading. Topics Christ Church 3 rd Year: Magnetic Resonance Reading The following sources are recommended for this tutorial: Nuclear Magnetic Resonance by P. J. Hore (Oxford Chemistry Primers). This text contains the

More information

2D NMR Spectroscopy: COSY Jessica Thomaston

2D NMR Spectroscopy: COSY Jessica Thomaston 2D NMR Spectroscopy: COSY Jessica Thomaston Outline 1. History 2. Theory of 2D NMR 3. Applications of COSY A brief history of 2D NMR In 1971, the idea of 2D NMR was proposed by Jean Jeener. The 1975 Ernst

More information

Introduction to NMR spectroscopy. Swiss Institute of Bioinformatics I.Phan & J. Kopp

Introduction to NMR spectroscopy. Swiss Institute of Bioinformatics I.Phan & J. Kopp Introduction to NMR spectroscopy Swiss Institute of Bioinformatics I.Phan & J. Kopp NMR: the background Complex technique. Requires knowledge in: Mathematics Physics Chemistry Biology (Medicin) Involves

More information

Chapter 13 Nuclear Magnetic Resonance Spectroscopy

Chapter 13 Nuclear Magnetic Resonance Spectroscopy Organic Chemistry, 6 th Edition L. G. Wade, Jr. Chapter 13 Nuclear Magnetic Resonance Spectroscopy Jo Blackburn Richland College, Dallas, TX Dallas County Community College District 2006, Prentice Hall

More information

Special versions of COSY can differentiate between short range and long range interactions, as illustrated below.

Special versions of COSY can differentiate between short range and long range interactions, as illustrated below. 2D NMR Spectroscopy To record a normal FT NMR spectrum we apply a pulse to our spin system and record the free induction decay (FID) following the pulse. The spectrum is obtained by Fourier Transform where

More information

Introduction to NMR Part 1. Revised 2/19/07 Anne M. Gorham

Introduction to NMR Part 1. Revised 2/19/07 Anne M. Gorham Introduction to NMR Part 1 Revised 2/19/07 Anne M. Gorham What is an NMR? Niobium-tin-copper clad coil wound like a spool of thread. The current runs through this coil, creating the magnetic field. This

More information

Electronic Supplementary Information

Electronic Supplementary Information Electronic Supplementary Material (ESI) for Physical Chemistry Chemical Physics. This journal is the Owner Societies 2016 Electronic Supplementary Information Achieving High Resolution and Controlling

More information

Nuclear Magnetic Resonance (NMR) Spectroscopy cont... Recommended Reading:

Nuclear Magnetic Resonance (NMR) Spectroscopy cont... Recommended Reading: Applied Spectroscopy Nuclear Magnetic Resonance (NMR) Spectroscopy cont... Recommended Reading: Banwell and McCash Chapter 7 Skoog, Holler Nieman Chapter 19 Atkins, Chapter 18 Relaxation processes We need

More information

By far the most important and useful technique to identify organic molecules. Often the only technique necessary.

By far the most important and useful technique to identify organic molecules. Often the only technique necessary. Chapter 13: NMR Spectroscopy 39 NMR Spectroscopy By far the most important and useful technique to identify organic molecules. Often the only technique necessary. NMR spectrum can be recorded for many

More information

Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance Spectroscopy Nuclear Magnetic Resonance Spectroscopy Nuclear magnetic resonance spectroscopy is a powerful analytical technique used to characterize organic molecules by identifying carbonhydrogen frameworks within

More information

NMR SPECTROSCOPY. Basic Principles, Concepts, and Applications in Chemistry. Harald Günther University of Siegen, Siegen, Germany.

NMR SPECTROSCOPY. Basic Principles, Concepts, and Applications in Chemistry. Harald Günther University of Siegen, Siegen, Germany. NMR SPECTROSCOPY Basic Principles, Concepts, and Applications in Chemistry Harald Günther University of Siegen, Siegen, Germany Second Edition Translated by Harald Günther JOHN WILEY & SONS Chichester

More information

Instrumental Lab. Nuclear Magnetic Resonance. Dr Alex J. Roche

Instrumental Lab. Nuclear Magnetic Resonance. Dr Alex J. Roche Instrumental Lab Nuclear Magnetic Resonance Dr Alex J. Roche 1 Nuclear Magnetic Resonance (NMR) Spectroscopy NMR is the most powerful analytical tool currently available to an organic chemist. NMR allows

More information

TWO DIMENSIONAL CORRELATED NMR SPECTROSCOPY

TWO DIMENSIONAL CORRELATED NMR SPECTROSCOPY TWO DIMENSIONAL 2D-NMR TWO DIMENSIONAL CORRELATED NMR SPECTROSCOPY Two-dimensional J-resolved spectrum: Frequencies plotted on one axis, coupling constants on the other. Two-dimensional shift correlated

More information

NMR for Organic Chemistry III

NMR for Organic Chemistry III NMR for rganic Chemistry III Lecture 1 Lecture 2 Lecture 3 Lecture 4 Recap of Key Themes from NMR II + Problems CSY + Problems HSQC + Problems HMBC and Solving Structures + Problems 1 1. Practical Aspects

More information

PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (H-NMR)

PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (H-NMR) PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (H-NMR) WHAT IS H-NMR SPECTROSCOPY? References: Bruice 14.1, 14.2 Introduction NMR or nuclear magnetic resonance spectroscopy is a technique used to determine

More information

A brief introduction to NMR spectroscopy of proteins. By Flemming M. Poulsen 2002

A brief introduction to NMR spectroscopy of proteins. By Flemming M. Poulsen 2002 A brief introduction to NMR spectroscopy of proteins By Flemming M. Poulsen 2002 1 Introduction Nuclear magnetic resonance, NMR, and X-ray crystallography are the only two methods that can be applied to

More information

NMR Experiments for Assignment. Dr Michael Thrippleton

NMR Experiments for Assignment. Dr Michael Thrippleton NMR Experiments for Assignment Dr Michael Thrippleton Introduction Selecting experiments for assignment: 1. what type of information do you want? chemical shifts coupling constants correlations 2. what

More information

4. It is possible to excite, or flip the nuclear magnetic vector from the α-state to the β-state by bridging the energy gap between the two. This is a

4. It is possible to excite, or flip the nuclear magnetic vector from the α-state to the β-state by bridging the energy gap between the two. This is a BASIC PRINCIPLES INTRODUCTION TO NUCLEAR MAGNETIC RESONANCE (NMR) 1. The nuclei of certain atoms with odd atomic number, and/or odd mass behave as spinning charges. The nucleus is the center of positive

More information

Structural Bioinformatics (C3210) Experimental Methods for Macromolecular Structure Determination

Structural Bioinformatics (C3210) Experimental Methods for Macromolecular Structure Determination Structural Bioinformatics (C3210) Experimental Methods for Macromolecular Structure Determination Introduction Knowing the exact 3D-structure of bio-molecules is essential for any attempt to understand

More information

Proton Nuclear Magnetic Resonance Spectroscopy

Proton Nuclear Magnetic Resonance Spectroscopy Proton Nuclear Magnetic Resonance Spectroscopy Introduction: The NMR Spectrum serves as a great resource in determining the structure of an organic compound by revealing the hydrogen and carbon skeleton.

More information

2D NMR Spectroscopy. Lecture 3

2D NMR Spectroscopy. Lecture 3 2D NMR Spectroscopy Lecture 3 hemical shifts The chemical environment affects the magnetic field of nuclei. B eff = B o - B loc B eff = B o ( - σ ) σ is the magnetic shielding of the nucleus. Factors that

More information

Determination of Equilibrium Constants using NMR Spectrscopy

Determination of Equilibrium Constants using NMR Spectrscopy CHEM 331L Physical Chemistry Laboratory Revision 1.0 Determination of Equilibrium Constants using NMR Spectrscopy In this laboratory exercise we will measure a chemical equilibrium constant using key proton

More information

The Four Questions to Ask While Interpreting Spectra. 1. How many different environments are there?

The Four Questions to Ask While Interpreting Spectra. 1. How many different environments are there? 1 H NMR Spectroscopy (#1c) The technique of 1 H NMR spectroscopy is central to organic chemistry and other fields involving analysis of organic chemicals, such as forensics and environmental science. It

More information

Nuclear Magnetic Resonance

Nuclear Magnetic Resonance Nuclear Magnetic Resonance NMR is probably the most useful and powerful technique for identifying and characterizing organic compounds. Felix Bloch and Edward Mills Purcell were awarded the 1952 Nobel

More information

BUILDING BLOCKS FOR MULTIDIMENSIONAL NMR AND SPECIAL CONSIDERATIONS FOR BIOLOGICAL APPLICATIONS OF NMR

BUILDING BLOCKS FOR MULTIDIMENSIONAL NMR AND SPECIAL CONSIDERATIONS FOR BIOLOGICAL APPLICATIONS OF NMR 09-Pochapsky-09-cpp 3/7/06 2:9 pm Page 24 9 BUILDING BLOCKS FOR MULTIDIMENSIONAL NMR AND SPECIAL CONSIDERATIONS FOR BIOLOGICAL APPLICATIONS OF NMR The development of multidimensional NMR (three or more

More information

Nuclear Magnetic Resonance (NMR) Spectroscopy

Nuclear Magnetic Resonance (NMR) Spectroscopy April 28, 2016 Exam #3: Graded exams on Tuesday! Final Exam Tuesday, May 10 th, 10:30 a.m. Room: Votey 207 (tentative) Review Session: Sunday, May 8 th, 4 pm, Kalkin 325 (tentative) Office Hours Next week:

More information

Exam Questions KJ 3022

Exam Questions KJ 3022 Exam Questions KJ 3022 Audun F. Buene audun.f.buene@gmail.com 1. desember 2013 These questions are answered by many contributors. If you find any errors, please let me know. Hopefully these short answers

More information

1. INTRODUCTION AND THEORY H- 1 H CORRELATION: COSY AND DQF-COSY EXPERIMENTS H- 1 H CORRELATION: TOCSY EXPERIMENT

1. INTRODUCTION AND THEORY H- 1 H CORRELATION: COSY AND DQF-COSY EXPERIMENTS H- 1 H CORRELATION: TOCSY EXPERIMENT ADVANCED NUCLEAR MAGNETIC RESONANCE (ANMR) UNIT 7 HOMONUCLEAR CORRELATION NMR SPECTROMETRY 2D-NMR 1. INTRODUCTION AND THEORY 2. 1 H- 1 H CORRELATION: COSY AND DQF-COSY EXPERIMENTS 3. 1 H- 1 H CORRELATION:

More information

Proton Nuclear Magnetic Resonance ( 1 H-NMR) Spectroscopy

Proton Nuclear Magnetic Resonance ( 1 H-NMR) Spectroscopy Proton Nuclear Magnetic Resonance ( 1 H-NMR) Spectroscopy Theory behind NMR: In the late 1940 s, physical chemists originally developed NMR spectroscopy to study different properties of atomic nuclei,

More information

Nuclear Magnetic Resonance

Nuclear Magnetic Resonance Nuclear Magnetic Resonance Introduction Atomic magnetism Nuclear magnetic resonance refers to the behaviour of atomic nuclei in the presence of a magnetic field. The first principle required to understand

More information

NMR Spectroscopy. Introduction

NMR Spectroscopy. Introduction Introduction NMR Spectroscopy Over the past fifty years nuclear magnetic resonance spectroscopy, commonly referred to as nmr, has become the most important technique for determining the structure of organic

More information

M z!=! N! 2! 2 I ( I +1)

M z!=! N! 2! 2 I ( I +1) Advantages and disadvantages of a high magnetic field NMR and 3 C detection 202 Jan. 25-26 th Akihabara Satellite campus of Tokyo Metropolitan Univ. The 3 th RRR-workshop 20/2 Institute for Protein Research

More information

Introduction to NMR Spectroscopy and Imaging Assignment for Chapter 02: Chemical shift and J Coupling

Introduction to NMR Spectroscopy and Imaging Assignment for Chapter 02: Chemical shift and J Coupling Introduction to NMR Spectroscopy and Imaging Assignment for Chapter 02: Chemical shift and J Coupling 0. Choose the correct one(s) from the following statements or explain briefly your supporting reason

More information

Two-dimensional NMR. Two-dimensional (2D) NMR spectroscopy was first proposed in 1971, but its full power was not realized until 1980.

Two-dimensional NMR. Two-dimensional (2D) NMR spectroscopy was first proposed in 1971, but its full power was not realized until 1980. Two-dimensional NMR Two-dimensional (D) NMR spectroscop was first proposed in 97, but its full power was not realied until 980. D pulse sequences consist of a preparation period (frequentl a 90 pulse),

More information

Protein Dynamics by NMR. Why NMR is the best!

Protein Dynamics by NMR. Why NMR is the best! Protein Dynamics by NMR Why NMR is the best! Key Points NMR dynamics divided into 2 regimes: fast and slow. How protein mobons affect NMR parameters depend on whether they are faster or slower than the

More information

Introduction to biomolecular NMR spectroscopy

Introduction to biomolecular NMR spectroscopy Introduction to biomolecular NMR spectroscopy Part I Michael Sattler, EMBL Heidelberg www.embl-heidelberg.de/nmr/sattler History Applications of NMR Principles of Nuclear Magnetic Resonance NMR observables

More information

Introductory to NMR Spectroscopy

Introductory to NMR Spectroscopy References: Introductory to NMR Spectroscopy Instructor: Tai-huang Huang ( ) Tel. (886)-2-2652-3036; E. mail: bmthh@ibms.sinica.edu.tw Web site: www.nmr.ibms.sinica.edu.tw/~thh/biophysics/nmr-2.ppt 1.

More information

Determining the Structure of an Organic Compound

Determining the Structure of an Organic Compound Determining the Structure of an Organic Compound The analysis of the outcome of a reaction requires that we know the full structure of the products as well as the reactants In the 19 th and early 20 th

More information

8.1 Relaxation in NMR Spectroscopy

8.1 Relaxation in NMR Spectroscopy 8.1 Relaxation in NMR Spectroscopy Copyright ans J. Reich 2010 All Rights Reserved University of Wisconsin An understanding of relaxation processes is important for the proper measurement and interpretation

More information

NMR practice times. Mo 2---8 pm Jim 2-4:30 Ning 4:30-7. Tues 2--- 8 pm Jianing 2-4:30 Ting 4:30-7. Fri 10- --- 7 Donia 10-12:00 Ilya 2-4

NMR practice times. Mo 2---8 pm Jim 2-4:30 Ning 4:30-7. Tues 2--- 8 pm Jianing 2-4:30 Ting 4:30-7. Fri 10- --- 7 Donia 10-12:00 Ilya 2-4 NMR practice times 1 Mo 2---8 pm Jim 2-4:30 Ning 4:30-7 Tues 2--- 8 pm Jianing 2-4:30 Ting 4:30-7 Wed 4:30---8 John 5:00-7:30 Fri 10- --- 7 Donia 10-12:00 Ilya 2-4 Samples are listed in the homework along

More information

The Hydrogen Atom Is a Magnet. http://www.seed.slb.com/en/scictr/watch/gashydrates/detecting.htm

The Hydrogen Atom Is a Magnet. http://www.seed.slb.com/en/scictr/watch/gashydrates/detecting.htm The Hydrogen Atom Is a Magnet Nuclear Magnetic Resonance Spectroscopy (NMR) Proton NMR A hydrogen nucleus can be viewed as a proton, which can be viewed as a spinning charge. As with any spinning charge,

More information

NMR Signal Properties & Data Processing

NMR Signal Properties & Data Processing COURSE#1022: Biochemical Applications of NMR Spectroscopy http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/ NMR Signal Properties & Data Processing LAST UPDATE: 1/13/2012 Reading Selected Readings

More information

1 H and 13 C NMR compared: Both give information about the number of chemically nonequivalent nuclei (nonequivalent

1 H and 13 C NMR compared: Both give information about the number of chemically nonequivalent nuclei (nonequivalent 1 H and 13 C NMR compared: 13 C NMR Spectroscopy Both give information about the number of chemically nonequivalent nuclei (nonequivalent hydrogens or nonequivalent carbons) Both give information about

More information

Spin-Lattice Relaxation Times

Spin-Lattice Relaxation Times Spin-Lattice Relaxation Times Reading Assignment: T. D. W. Claridge, High Resolution NMR Techniques in Organic Chemistry, Chapter 2; E. Breitmaier, W. Voelter, Carbon 13 NMR Spectroscopy,3rd Ed., 3.3.2.

More information

Spectroscopy. energy Low λ High ν. UV-visible

Spectroscopy. energy Low λ High ν. UV-visible Spectroscopy frequency 10 20 10 18 10 16 10 14 10 12 10 8 Gamma rays X-rays UV IR Microwaves Radiowaves High energy Low λ High ν visible Low energy quantization of energy levels X-Ray UV-visible Infrared

More information

NMR Spectroscopy: Principles and Applications. Nagarajan Murali Basic Concepts Lecture 1

NMR Spectroscopy: Principles and Applications. Nagarajan Murali Basic Concepts Lecture 1 NMR Spectroscopy: Principles and Applications Nagarajan Murali Basic Concepts Lecture 1 NMR Spectroscopy: Principles and Applications (16:160:542 Cross Listed 01:160:488:03) Instructors: Prof. Jean Baum

More information

Chapter 15 NMR Spectroscopy

Chapter 15 NMR Spectroscopy Chempocalypse Now! Chapter 15 NMR Spectroscopy Page 1 Chapter 15 NMR Spectroscopy Parts of Topics A5 and A9 from the IB HL Chemistry Curriculum A5 A.5.1 Nuclear magnetic resonance (NMR) spectrometry (2

More information

Chemical shift = observed chemical shift in MHz/ frequency of spectrometer (MHz)

Chemical shift = observed chemical shift in MHz/ frequency of spectrometer (MHz) Chapter 4. Physical Basis of NMR Spectroscopy. Today the most widely used method for determining the structure of organic compounds is nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy involves

More information

Basic Principles of Magnetic Resonance

Basic Principles of Magnetic Resonance Basic Principles of Magnetic Resonance Contents: Jorge Jovicich jovicich@mit.edu I) Historical Background II) An MR experiment - Overview - Can we scan the subject? - The subject goes into the magnet -

More information

Chapter 16: NMR Spectroscopy (i.e., the most exciting thing on the planet)

Chapter 16: NMR Spectroscopy (i.e., the most exciting thing on the planet) Chapter 16: NMR Spectroscopy (i.e., the most exciting thing on the planet) ne day (soon), the following will make a lot of sense to you: 16.1-16.3 The Proton: An Unexpected Journey NMR: Like electrons,

More information

Used to determine relative location of atoms within a molecule Most helpful spectroscopic technique in organic chemistry Related to MRI in medicine

Used to determine relative location of atoms within a molecule Most helpful spectroscopic technique in organic chemistry Related to MRI in medicine Structure Determination: Nuclear Magnetic Resonance CHEM 241 UNIT 5C 1 The Use of NMR Spectroscopy Used to determine relative location of atoms within a molecule Most helpful spectroscopic technique in

More information

Nuclear Magnetic Resonance notes

Nuclear Magnetic Resonance notes Reminder: These notes are meant to supplement, not replace, the laboratory manual. Nuclear Magnetic Resonance notes Nuclear Magnetic Resonance (NMR) is a spectrometric technique which provides information

More information

H NMR (proton NMR): determines number and type of H atoms 13. C NMR (proton NMR): determines number and type of C atoms

H NMR (proton NMR): determines number and type of H atoms 13. C NMR (proton NMR): determines number and type of C atoms 14.1 An Introduction to NMR Spectroscopy A. The Basics of Nuclear Magnetic Resonance (NMR) Spectroscopy nuclei with odd atomic number have a S = ½ with two spin states (+1/2 and -1/2) 1 H NMR (proton NMR):

More information

Chapter 11 Structure Determination: Nuclear Magnetic Resonance Spectroscopy. Nuclear Magnetic Resonance Spectroscopy. 11.1 Nuclear Magnetic Resonance

Chapter 11 Structure Determination: Nuclear Magnetic Resonance Spectroscopy. Nuclear Magnetic Resonance Spectroscopy. 11.1 Nuclear Magnetic Resonance John E. McMurry http://www.cengage.com/chemistry/mcmurry Chapter 11 Structure Determination: Nuclear Magnetic Resonance Spectroscopy 11.1 Nuclear Magnetic Resonance Spectroscopy Many atomic nuclei behave

More information

Proton NMR. One Dimensional H-NMR. Cl S. Common types of NMR experiments: 1-H NMR

Proton NMR. One Dimensional H-NMR. Cl S. Common types of NMR experiments: 1-H NMR Common types of NMR experiments: 1- NMR Proton NMR ne Dimensional -NMR a. Experiment igh field proton NMR (400Mz). single-pulse experiment. b. Spectral nterpretation i. Number of multiplets gives the different

More information

Nuclear Magnetic Resonance Spectroscopy (NMR)

Nuclear Magnetic Resonance Spectroscopy (NMR) Nuclear Magnetic Resonance Spectroscopy (NMR) NMR is a spectroscopic technique which relies on the magnetic properties of the atomic nucleus. When placed in a strong magnetic field, certain nuclei resonate

More information

Chapter 13 Spectroscopy NMR, IR, MS, UV-Vis

Chapter 13 Spectroscopy NMR, IR, MS, UV-Vis Chapter 13 Spectroscopy NMR, IR, MS, UV-Vis Main points of the chapter 1. Hydrogen Nuclear Magnetic Resonance a. Splitting or coupling (what s next to what) b. Chemical shifts (what type is it) c. Integration

More information

April 24, 2015. A Classical Perspective. Exam #3: Solution Key online now! Graded exams by Monday!

April 24, 2015. A Classical Perspective. Exam #3: Solution Key online now! Graded exams by Monday! April 24, 2015 Exam #3: Solution Key online now! Graded exams by Monday! Final Exam Monday, May 4 th, 10:30 a.m. Room: Perkins 107 1 A Classical Perspective A classical view will help us understand the

More information

NMR Guidelines for ACS Journals

NMR Guidelines for ACS Journals NMR Guidelines for ACS Journals Updated December 2013 1. NMR Text (Experimental Section) 1.1 The compound must be clearly identified, for example in a header at the beginning of a) the synthetic procedure

More information

NMR SPECTROSCOPY A N I N T R O D U C T I O N T O... Self-study booklet NUCLEAR MAGNETIC RESONANCE. 4 3 2 1 0 δ PUBLISHING

NMR SPECTROSCOPY A N I N T R O D U C T I O N T O... Self-study booklet NUCLEAR MAGNETIC RESONANCE. 4 3 2 1 0 δ PUBLISHING A N I N T R O D U T I O N T O... NMR SPETROSOPY NULEAR MAGNETI RESONANE 4 3 1 0 δ Self-study booklet PUBLISING NMR Spectroscopy NULEAR MAGNETI RESONANE SPETROSOPY Origin of Spectra Theory All nuclei possess

More information

Interpretation of Experimental Data

Interpretation of Experimental Data Lab References When evaluating experimental data it is important to recognize what the data you are collecting is telling you, as well as the strengths and limitations of each method you are using. Additionally,

More information

Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance Spectroscopy Most spinning nuclei behave like magnets. Nuclear Magnetic Resonance Spectroscopy asics owever, as opposed to the behavior of a classical magnet the nuclear spin magnetic moment does not always align with

More information

Determination of Molecular Structure by MOLECULAR SPECTROSCOPY

Determination of Molecular Structure by MOLECULAR SPECTROSCOPY Determination of Molecular Structure by MOLEULAR SPETROSOPY hemistry 3 B.Z. Shakhashiri Fall 29 Much of what we know about molecular structure has been learned by observing and analyzing how electromagnetic

More information

Infrared Spectroscopy 紅 外 線 光 譜 儀

Infrared Spectroscopy 紅 外 線 光 譜 儀 Infrared Spectroscopy 紅 外 線 光 譜 儀 Introduction Spectroscopy is an analytical technique which helps determine structure. It destroys little or no sample (nondestructive method). The amount of light absorbed

More information

Nuclear Magnetic Resonance (NMR) Analysis of D - (+) - Glucose: A Guide to Spectrometric Structural Elucidation of Sugars

Nuclear Magnetic Resonance (NMR) Analysis of D - (+) - Glucose: A Guide to Spectrometric Structural Elucidation of Sugars IOSR Journal of Applied Chemistry (IOSR-JAC) e-issn: 2278-5736.Volume 6, Issue 1 (Nov. Dec. 2013), PP 45-51 Nuclear Magnetic Resonance (NMR) Analysis of D - (+) - Glucose: A Guide to Spectrometric Structural

More information

Biophysical Chemistry: NMR Spectroscopy

Biophysical Chemistry: NMR Spectroscopy General Principle The Chemical Shift Vrije Universiteit Brussel 28th October 2011 General Principle Outline 1 General Principle Influence of Electron Clouds 2 3 Outline General Principle Influence of Electron

More information

Practical guide for quantitative 1D NMR integration Eugenio Alvarado, University of Michigan, 05/10/10

Practical guide for quantitative 1D NMR integration Eugenio Alvarado, University of Michigan, 05/10/10 Practical guide for quantitative 1D NMR integration Eugenio Alvarado, University of Michigan, 05/10/10 The purpose of this manuscript is not to present a discussion about quantitative NMR, but to offer

More information

Chapter 19 Nuclear Magnetic Resonance Spectroscopy (NMR)

Chapter 19 Nuclear Magnetic Resonance Spectroscopy (NMR) Chapter 19 Nuclear Magnetic Resonance Spectroscopy (NMR) A shorter version of the notes, designed to be covered in 4 days. Problems : 1, 2, 3, 4, 7, 10, 11, 19, 20, 22, 24, 27, 30, 34, 35 Absorption of

More information

Background A nucleus with an odd atomic number or an odd mass number has a nuclear spin that can be observed by NMR spectrometers.

Background A nucleus with an odd atomic number or an odd mass number has a nuclear spin that can be observed by NMR spectrometers. NMR Spectroscopy I Reading: Wade chapter, sections -- -7 Study Problems: -, -7 Key oncepts and Skills: Given an structure, determine which protons are equivalent and which are nonequivalent, predict the

More information

Introduction to Protein Structure Bioinformatics 2004 NMR Spectroscopy. CH.EMBnet course Biozentrum, Basel. D. Häussinger.

Introduction to Protein Structure Bioinformatics 2004 NMR Spectroscopy. CH.EMBnet course Biozentrum, Basel. D. Häussinger. Introduction to Protein Structure Bioinformatics 2004 NMR Spectroscopy CH.EMBnet course 28.9.2004 Biozentrum, Basel D. Häussinger Overview 1. Basic principles of NMR 2. Structure Determination by Solution

More information

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY PRINCIPLE AND APPLICATION IN STRUCTURE ELUCIDATION Professor S. SANKARARAMAN Department of Chemistry Indian Institute of Technology Madras Chennai 600 036 sanka@iitm.ac.in

More information

Yin and Yang in Chemistry Education: The Complementary Nature of FT-IR and NMR Spectroscopies

Yin and Yang in Chemistry Education: The Complementary Nature of FT-IR and NMR Spectroscopies Yin and Yang in Chemistry Education: The Complementary Nature of FT-IR and NMR Spectroscopies Matthew Gundlach, Katherine Paulsen, Michael Garry, Steve Lowry, Thermo Fisher Scientific, Madison, WI USA

More information