1 NMR Spectroscopy: Introduction – History of NMR Introduction – History of NMR NMR Hardware and Software NMR Hardware and Software Superconducting Cryo-Magnet

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NMR NMR Spectroscopy:Spectroscopy:

Introduction Introduction –– History of NMR History of NMR NMR Hardware and SoftwareNMR Hardware and Software

Superconducting Cryo-MagnetSuperconducting Cryo-Magnet ProbeProbe RF ConsoleRF Console Computer + NMR Software Computer + NMR Software

+ Printer/Plotter + Printer/Plotter Solution NMR Solution NMR

Sample PreparationSample Preparation Presentation of DataPresentation of Data

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Important NMR MilestonesImportant NMR Milestones

1938 - NMR in molecular beams 1938 - NMR in molecular beams Rabi (Columbia University)Rabi (Columbia University)

1946 - NMR of Liquids and Solids1946 - NMR of Liquids and Solids Purcell, Torrey, Pound (Harvard)Purcell, Torrey, Pound (Harvard) Bloch, Hansen, Packard (CalTech)Bloch, Hansen, Packard (CalTech)

1952 - First commercial NMR spectrometer1952 - First commercial NMR spectrometer 1962 - First Superconducting Magnet for 1962 - First Superconducting Magnet for

NMRNMR 1968 - First Pulse Fourier Transform NMR1968 - First Pulse Fourier Transform NMR 1969 - First Concept of MRI Scanners1969 - First Concept of MRI Scanners 1971 - First 2D NMR Experiment – COSY 1971 - First 2D NMR Experiment – COSY

(Jean Jeener)(Jean Jeener) 1985 - Protein Structures1985 - Protein Structures 2009 - First Gigahertz NMR Spectrometer2009 - First Gigahertz NMR Spectrometer

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NMR Nobel Prize NMR Nobel Prize WinnersWinners

1944 Isador Rabi1944 Isador Rabi 1952 Felix Bloch 1952 Felix Bloch

& Edwin Purcell& Edwin Purcell

1991 Richard Ernst1991 Richard Ernst 2002 Kurt Wüthrich2002 Kurt Wüthrich

2003 Paul Lauterbur 2003 Paul Lauterbur & Sir Peter & Sir Peter

MansfieldMansfield

From: Bruker SpinReport, Vol 153

Laukien Prize WinnersLaukien Prize Winners

1999 Konstantin Pervushin, Roland Riek, Gerhard Wider, and Kurt Wüthrich; 1999 Konstantin Pervushin, Roland Riek, Gerhard Wider, and Kurt Wüthrich; TROSYTROSY 2000 Lucio Frydman; 2000 Lucio Frydman; Quadrupolar MQMASQuadrupolar MQMAS 2001 Peter Boesiger, Klaas Prüßmann, Markus Weiger; 2001 Peter Boesiger, Klaas Prüßmann, Markus Weiger; Sensitivity-encoded magnetic Sensitivity-encoded magnetic

resonance imagingresonance imaging 2002 Ad Bax, Aksel Bothner-By and James Prestegard; 2002 Ad Bax, Aksel Bothner-By and James Prestegard; Residual dipolar couplings of weakly Residual dipolar couplings of weakly

aligned molecules in solutionaligned molecules in solution 2003 Jacob Schaefer; 2003 Jacob Schaefer; REDOR Technique for Solid State NMRREDOR Technique for Solid State NMR 2004 Lewis E. Kay, 2004 Lewis E. Kay, NMR of Biological MacromoleculesNMR of Biological Macromolecules 2005 Stephan Grzesiek, 2005 Stephan Grzesiek, J couplings across hydrogen J couplings across hydrogen

bondsbonds 2006 Thomas Szyperski, Eriks Kupce, Ray Freeman, and Rafael Bruschweiler; 2006 Thomas Szyperski, Eriks Kupce, Ray Freeman, and Rafael Bruschweiler; Acceleration of Multi-Acceleration of Multi-

dimensional NMR dimensional NMR by novel procedures for scanning data space and efficiently processing results to obtain a conventional by novel procedures for scanning data space and efficiently processing results to obtain a conventional

spectral representationspectral representation 2007 Robert G. Griffin; 2007 Robert G. Griffin; High-field dynamic nuclear polarization (DNP) for sensitivity enhancement in High-field dynamic nuclear polarization (DNP) for sensitivity enhancement in

solid-state MAS NMRsolid-state MAS NMR 2008 Malcom H. Levitt; 2008 Malcom H. Levitt; Optimized pulses and pulse sequences to enhance the power of Optimized pulses and pulse sequences to enhance the power of

liquid & solid state NMRliquid & solid state NMR 2009 Daniel P. Weitekamp; 2009 Daniel P. Weitekamp; PASADENA and BOOMERANG PASADENA and BOOMERANG

significantly improve NMR force detection by circumventing the problems of significantly improve NMR force detection by circumventing the problems of inhomogeneous magnetic fieldsinhomogeneous magnetic fields

2010 Paul T. Callahan; 2010 Paul T. Callahan; Contributions to the study of polymeric and heterogeneous materials by advanced NMR exchange, diffusion and Contributions to the study of polymeric and heterogeneous materials by advanced NMR exchange, diffusion and relaxation techniques, and for his innovative q-space-diffusion-related developments that were relevant in the relaxation techniques, and for his innovative q-space-diffusion-related developments that were relevant in the context of the development of diffusion-tensor imaging.context of the development of diffusion-tensor imaging.

2011 Daniel Rugar, John Mamin, and John Sidles; 2011 Daniel Rugar, John Mamin, and John Sidles; Magnetic Resonance Force Magnetic Resonance Force Microscopy (MRFM). Microscopy (MRFM).

2012 Klaes Golman and Jan Henrik Ardenkjaer-Larsen: 2012 Klaes Golman and Jan Henrik Ardenkjaer-Larsen: Dissolution-DNP NMR Dissolution-DNP NMR 4

Varian Prize WinnersVarian Prize Winners

20122012 Ray Freeman and Weston A. AndersonRay Freeman and Weston A. Anderson Nuclear Nuclear Magnetic Double ResonanceMagnetic Double Resonance

20112011 Gareth Alun Morris, The University of Manchester, UKGareth Alun Morris, The University of Manchester, UK INEPTINEPT 20102010 Martin Karplus, Harvard University, Cambridge, MassachusettsMartin Karplus, Harvard University, Cambridge, Massachusetts Karplus Karplus

equationsequations 20092009 Albert W. Overhauser, Purdue University, West Lafayette, INAlbert W. Overhauser, Purdue University, West Lafayette, IN NOE NOE

& Dynamic Polarization& Dynamic Polarization 20082008 Alexander Pines, UC Berkeley, and Lawrence Berkeley National LaboratoryAlexander Pines, UC Berkeley, and Lawrence Berkeley National Laboratory

Cross PolarizationCross Polarization 20072007 Alfred G. Redfield, Brandeis University, Waltham, MassachusettsAlfred G. Redfield, Brandeis University, Waltham, Massachusetts Spin DynamicsSpin Dynamics 20062006 John S. Waugh, MIT, Cambridge, MassachusettsJohn S. Waugh, MIT, Cambridge, Massachusetts Average Average

Hamiltonian Theory (AHT) Hamiltonian Theory (AHT) 20052005 Nicolaas Bloembergen, University of Arizona, Tucson, ArizonaNicolaas Bloembergen, University of Arizona, Tucson, Arizona Nuclear Nuclear

Magnetic RelaxationMagnetic Relaxation 20042004 Erwin L. Hahn, Professor Emeritus, University of California, BerkeleyErwin L. Hahn, Professor Emeritus, University of California, Berkeley Spin EchoesSpin Echoes 20022002 Jean Jeener, Universite Libre de Bruxelles, BelgiumJean Jeener, Universite Libre de Bruxelles, Belgium Two-Two-

dimensional NMR dimensional NMR

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NMR SpectroscopyNMR Spectroscopy

NNUCLEARUCLEAR

MMAGNETICAGNETIC

RRESONANCEESONANCE

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Superconducting Cryo-Superconducting Cryo-MagnetMagnet

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Superconducting Cryo-Superconducting Cryo-MagnetMagnet

superconducting superconducting wirewire

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NMR Magnet SafetyNMR Magnet Safety NMR magnets are always charged!NMR magnets are always charged!

NMR magnets may interfere with medical NMR magnets may interfere with medical devices (i.e. pacemakers, insulin pumps)devices (i.e. pacemakers, insulin pumps)

NMR magnets will erase credit cards, ID cards, NMR magnets will erase credit cards, ID cards, floppy disks, hard disks (some mp3 players).floppy disks, hard disks (some mp3 players).

NMR magnets and RF consoles may interfere with electronic and NMR magnets and RF consoles may interfere with electronic and mechanical devices and may damage them (cell phones, pagers, mechanical devices and may damage them (cell phones, pagers, watches, I-pods, etc.)watches, I-pods, etc.)

NMR magnets will attract ferromagnetic objects of any size (i.e. paper NMR magnets will attract ferromagnetic objects of any size (i.e. paper clips, coins, keys, pens, scissors, screw drivers, wrenches, metallic clips, coins, keys, pens, scissors, screw drivers, wrenches, metallic chairs, gas cylinders, etc.) and spectrometer and people may sustain chairs, gas cylinders, etc.) and spectrometer and people may sustain severe damage or injury, if handled carelessly.severe damage or injury, if handled carelessly.

NMR magnets contain Cryogens (liquid Helium and Nitrogen)NMR magnets contain Cryogens (liquid Helium and Nitrogen) Cryogens can cause severe burns if handled improperly Cryogens can cause severe burns if handled improperly

(use eye protection and gloves during refills).(use eye protection and gloves during refills). Cryogens evaporate and may cause asphyxiation if a lab is not properly Cryogens evaporate and may cause asphyxiation if a lab is not properly

ventilated.ventilated. During a magnet quench up to 100 liters of liquid Helium are vaporized During a magnet quench up to 100 liters of liquid Helium are vaporized

in a matter of minutes (2600 cu ft, 70,000 liters gas) and may cause in a matter of minutes (2600 cu ft, 70,000 liters gas) and may cause asphyxiation, even if the lab is well ventilated. If a magnet quenches, asphyxiation, even if the lab is well ventilated. If a magnet quenches, leave the lab immediately. leave the lab immediately. Don’t panic, helium gas will rise to the ceiling and escape through Don’t panic, helium gas will rise to the ceiling and escape through cracks.cracks.

During a refill the refill rubber tubing may shatter. Frozen rubber cuts During a refill the refill rubber tubing may shatter. Frozen rubber cuts like glass!!like glass!!

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NMR Magnet SafetyNMR Magnet Safety NMR magnets are always charged!NMR magnets are always charged!

NMR magnets contain Cryogens (liquid Helium and NMR magnets contain Cryogens (liquid Helium and Nitrogen)Nitrogen)

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NMR Console with NMR Console with ComputerComputer

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RF Signal GeneratorRF Signal Generator

Decoupler Decoupler ((11H):H):

AmplifierAmplifier

Frequency Frequency GeneratorGenerator

Transmitter:Transmitter:AmplifierAmplifier

Frequency Frequency GeneratorGenerator

Frequency Generators and Signal Amplifiers are required for each RF channel. Our spectrometers have 2 channels, modern spectrometers can have up to 8 channels.

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NMR NMR ProbesProbes

Solids Liquids

Solids

Liquids

Magic angle(54.7°)

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NMR Signal NMR Signal GenerationGenerationSpectrometer:Spectrometer:

RF Generation:RF Generation:

90°90° 180 °180 °Pulse (Sequences):Pulse (Sequences): RD RD ττ ττ DEDE AQ AQ

Receiver:Receiver: FTFT

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NMR SamplesNMR Samples

Types of NMR sample holdersSample preparationSpectrum quality

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Types of NMR Sample Types of NMR Sample HoldersHolders

Solid State Sample Rotors

Solution NMR Sample Tube Spinners

NMR Sample Tubes with Caps

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NMR Sample NMR Sample PreparationPreparationTubes and Caps:Tubes and Caps:

NMR tubes are a standard length (7 and 9 inch). When chipped (and NMR tubes are a standard length (7 and 9 inch). When chipped (and reduced in length) they should not be reused as an unbalanced tube reduced in length) they should not be reused as an unbalanced tube will not spin. will not spin.

Always clean the tubes thoroughly after use. Always clean the tubes thoroughly after use. First use the solvent you were using to recover your previous sample, First use the solvent you were using to recover your previous sample, then rinse several times with acetone and finally dry the sample tube then rinse several times with acetone and finally dry the sample tube laying flat on a layer of kimwipes or placed upside-down on a kimwipe laying flat on a layer of kimwipes or placed upside-down on a kimwipe in a beaker or Erlenmeyer flask. Choose the container so the tubes in a beaker or Erlenmeyer flask. Choose the container so the tubes stand vertically. Don’t heat the tubes above 50 stand vertically. Don’t heat the tubes above 50 °°C, as the glass might C, as the glass might warp. warp. Always store unused, clean tubes uncapped and laying on a flat surface.Always store unused, clean tubes uncapped and laying on a flat surface.

Tube caps are disposable and replacements can be easily obtained in Tube caps are disposable and replacements can be easily obtained in bags of 100 ($5) or 1000 ($40 at www.wilmad.com). bags of 100 ($5) or 1000 ($40 at www.wilmad.com).

Degassing Samples:Degassing Samples:

NMR spectra recorded using degassed solvents usually benefit from NMR spectra recorded using degassed solvents usually benefit from reduced half-height line-width and thus better S/N. (Oreduced half-height line-width and thus better S/N. (O22 gas is gas is paramagnetic!)paramagnetic!)

There are several ways of degassing your sample: There are several ways of degassing your sample: the best is the freeze-pump-thaw technique, the best is the freeze-pump-thaw technique, placing the sample in a ultrasonic bath works moderately well, placing the sample in a ultrasonic bath works moderately well, bubbling nitrogen through or over the sample less well. bubbling nitrogen through or over the sample less well.

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NMR Sample NMR Sample PreparationPreparation

Quantity:Quantity:

For proton NMR spectra of small organic compounds (up to MW=500) For proton NMR spectra of small organic compounds (up to MW=500) anything between 1 and 20 mg of sample will be fine. anything between 1 and 20 mg of sample will be fine. Concentrated solutions can be viscous and may result in broad signals. Concentrated solutions can be viscous and may result in broad signals. Very dilute samples could be masked by impurities and solvent peaks. Very dilute samples could be masked by impurities and solvent peaks.

Carbon-13 is present at approximately 1.1 % natural abundance. Carbon-13 is present at approximately 1.1 % natural abundance. It is intrinsically less sensitive than protons (approx. It is intrinsically less sensitive than protons (approx. six thousand six thousand times)times). . Please provide as much sample as possible, 50 - 100 mg (or more) is fine. Please provide as much sample as possible, 50 - 100 mg (or more) is fine. Preparing two samples - one dilute sample for proton NMR and one Preparing two samples - one dilute sample for proton NMR and one concentrated sample for carbon NMR is a useful, but unnecessary concentrated sample for carbon NMR is a useful, but unnecessary practice. practice.

Solvent height (volume) should be uniform, 5 cm or 2 inches equal 0.5 Solvent height (volume) should be uniform, 5 cm or 2 inches equal 0.5 ml. ml. The ends of the sample distort the field homogeneity, shimming on each The ends of the sample distort the field homogeneity, shimming on each sample corrects this effect and takes just a minute or so. However, vastly sample corrects this effect and takes just a minute or so. However, vastly different solvent heights (volumes) prevent complete correction and different solvent heights (volumes) prevent complete correction and require many minutes shimming to achieve acceptable homogeneity. require many minutes shimming to achieve acceptable homogeneity.

Samples prepared with too much solvent waste both time and money, Samples prepared with too much solvent waste both time and money, and provide poorer S/N. However, If you have limited amounts of sample and provide poorer S/N. However, If you have limited amounts of sample (less than 1 mg), using less solvent is permissible. (less than 1 mg), using less solvent is permissible. Minimum height: 1 cm, however, this requires special positioning of the Minimum height: 1 cm, however, this requires special positioning of the sample tube and very intensive shimming. sample tube and very intensive shimming.

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• Use clean + dry NMR tubes and caps (tubes can be re-used, caps should not!)

• 0.5 ml deuterated solvent (i.e. CDCl ,C D , acetone-d ,etc.)

• substrate requirements for routine spectra: 10 mg for proton NMR 100 mg for carbon-13 NMR

• min. filling height of tube: 2 inches (5 cm)

• Cleaning of tubes: 1. rinse with solvent you were using 2. rinse with acetone 3. dry in (vacuum-)oven at low temperature

3 6 6 6

5 mm

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Clean clear

solution

Suspension or opaque solution

PrecipitateNot

enough solvent

Two phases

Concentration gradient

GOOD! B a d S a m p l e s !

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Shimming

improves the magnetic field homogeneity

If the magnetic field is not uniform within the sample, molecules in different positions will experience different field strengths.

This will produce broad, distorted, or additional signals.

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Good and bad NMR Good and bad NMR SpectraSpectra

… are the result of:

Homogeneity of magnetic fieldSample preparationChoice of solventData acquisition parametersProcessing procedures

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Good spectrumGood spectrum ppm

ppm

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Good spectrumGood spectrum

Integrals

Peak picking

ppm scaleppm

ppm

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Good spectrumGood spectrum

ppm

ppm

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Bad spectrum ?Bad spectrum ?

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Bad spectrum !Bad spectrum !

Signal/Noise ratio bad

No units specified for axis and peak picking

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Tall signals are cut off

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Signals too small

(only allowed when trying to compare signal intensities between

different spectra)

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Broad signals Possible reasons:

poor shimmingviscous samplesample too concentratedsuspended particles in sample

excessive line broadening may have been used during processing

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Signals are distorted

(automatic phase correction is often insufficient)

Excessive peak picking

(low p.p. threshold,also due to improper phasing)

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Areas without signals should be excluded.

(If you want to print all your spectra with a default range, i.e. 0-10 ppm, don’t forget to

print detailed expansions.)

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Lab AssignmentsLab Assignments

1. 3x2 hrs x x x Comprehensive 1H NMR and Indirect 13C Observation 25%

2. 2x2 hrs x x Pulse Width Calibration and B1 100 mg

3. 2x2 hrs x x Rare spin NMR (13C, 17O, 29Si) 80%

4. 3 hrs x Decoupling 1H from 13C and B2 100 mg

5. 2 hrs x Spin Echo and Spin/Spin Relaxation, 13C - T2 100 mg

6. 2 hrs x x Spin/Lattice Relaxation, 13C - T1 100 mg

7. 3 hrs x x Nuclear Overhauser Effect 10 mg

8. 2 hrs x Polarization Transfer and DEPT 10 mg 9. 3 hrs d x 13C CP/MAS in Solids (sample

provided)10. 2 hrs x HETCOR (1H/13C HSQC) 10 mg11. 2 hrs x 1H COSY / NOESY 25 mg

Use your own samples, if possible. Familiarize yourselves with the procedures for each experiment, before you come to the lab. Print the manuals.

http://nmr.binghamton.edu

Instrument:600 300 100

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In all assignments you are expected to report on the following In all assignments you are expected to report on the following information:information:

Signal-to-noise ratioSignal-to-noise ratio Chemical shifts and method of reference in relation to structureChemical shifts and method of reference in relation to structure J coupling (homo and heteronuclear) in relation to structureJ coupling (homo and heteronuclear) in relation to structure Peak intensity and area in relation to concentration and Peak intensity and area in relation to concentration and

structure/dynamics/relaxationstructure/dynamics/relaxation Effect of B0 / lab magnetic field strength, Larmor frequency and Effect of B0 / lab magnetic field strength, Larmor frequency and

gyromagnetic ratiogyromagnetic ratio Effect of B0 inhomogeneityEffect of B0 inhomogeneity Pulse width and tip anglePulse width and tip angle Magnitude of B1 and B2 fields / rf field strengths used Magnitude of B1 and B2 fields / rf field strengths used Effect of B1 and B2 inhomogeneityEffect of B1 and B2 inhomogeneity Effects of O1 and O2 / frequency offsetsEffects of O1 and O2 / frequency offsets Effects of sample spinning and location of spinning sidebandsEffects of sample spinning and location of spinning sidebands Method of decoupling if used as well as effectivenessMethod of decoupling if used as well as effectiveness Solvent and temperature usedSolvent and temperature used Pulse sequence and instrument control programmingPulse sequence and instrument control programming All critical instrument or data processing parametersAll critical instrument or data processing parameters Vector and spin diagrams, as needed. Vector and spin diagrams, as needed. Note any experimental or instrumental anomaliesNote any experimental or instrumental anomalies

The spectrometer manual to be used in this course can be downloaded from:The spectrometer manual to be used in this course can be downloaded from:http://www.chem.binghamton.edu/staff/schulte/CHEM585f/Chem585f-Bruker.doc

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A letter in the New Scientist of 17 April 1999, signed by Terry McStea, Whitburn, Tyne and Wear: A letter in the New Scientist of 17 April 1999, signed by Terry McStea, Whitburn, Tyne and Wear:

Your article on MRI (This Week, 3 April, p 7) reminded me of a story, probably untrue, related by a Your article on MRI (This Week, 3 April, p 7) reminded me of a story, probably untrue, related by a doctor friend. doctor friend.

MRI used to be known in hospitals as nuclear magnetic resonance, or NMR. MRI used to be known in hospitals as nuclear magnetic resonance, or NMR.

Unfortunately, patients who arrived at the hospital asking for "an NMR" often received a treatment Unfortunately, patients who arrived at the hospital asking for "an NMR" often received a treatment that they were not expecting. that they were not expecting.

Hence the change to MRI. Hence the change to MRI.

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Documents

1 NMR Spectroscopy: Introduction – History of NMR Introduction – History of NMR NMR Hardware and Software NMR Hardware and Software Superconducting Cryo-Magnet