NMR: PRACTICAL ASPECTS

NMR: PRACTICAL ASPECTSPedro M. Aguiar

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Sample Preparation• Well prepared sample can yield high quality spectra • Poorly prepared sample typically yields low quality spectra

• Tubes of appropriate quality • Higher fields require higher quality tubes • Contact local facility manager for specifics

• Sample conc. at typical field instruments (MWt. < 1000) • 1H NMR: >= 10 mM

• 13C NMR: > = 40 mM

• Filter sample to remove insoluble material • No signals • Hampers ability to detect signal of soluble components

• Dry using vacuum and store in ovens no warmer than 80 °C

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Sample PreparationAssumed detection region for shimming

ideal

reality

Good 35-45 mm

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ideal

reality

• Poor shimming • Weak signal (not in

detector)

Short < 35 mm

Good 35-45 mm

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ideal

reality

• Poor shimming • Weak signal (not in

detector)

• May impact shimming • Weak signal (dilute)

Short < 35 mm

Good 35-45 mm

Long > 45 mm

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Sample Volume: 5 mm NMR tube

600 µL volume Well-shimmed Good lineshape

300 µL volume poorly-shimmed Poor lineshape

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Limited Sample volume• In cases of Limited sample volume partially filled 5 mm tube

yields poor data

• Alternatives

• Smaller NMR tubes • 3mm tubes (180-200 µL volume)

• Coaxial Inserts • 50-200 µL volumes

• Susceptibility-matched (Shigemi) tubes • 80-300 µL

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Relative Chemical Shifts

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Downfield

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UpfieldDownfield

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UpfieldDownfield

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UpfieldDownfield

Lower frequencyHigher frequency

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UpfieldDownfield


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✓

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Shielded or More shielded

De-shielded or Less shielded

UpfieldDownfield


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✓

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Shielded or More shielded

De-shielded or Less shielded

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Apodisation and Processing

FT

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Exponential Apodisation (line broadening) is most common for 1D

Optimum is to use “LB” of 0.5-1 times FWHM of peaks

€

e−πLBt

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FT

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Quantitative NMR?• The integrated signal intensity in NMR can be reflective of the

number of nuclei of a given type

1.01.52.02.53.03.5 ppm

3.0

2.0

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Quantitative NMR?• The integrated signal intensity in NMR can be reflective of the

number of nuclei of a given type

• Caveats • Signal intensities not enhanced artificially (e.g., nOe, INEPT/DEPT, pH2)

• Often 13C, 31P, 11B and 29Si on walk-up instruments not quantitative

• All nuclei must be allowed to reach equilibrium before spectrum acquired • Pre-experiment/recycle/relaxation delay must be long enough • 3-5 times T1

• 1H (1-10s), 19F (1-20 s), 13C (10-600 s), 29Si (5-1000 s)

rd

rd

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Quantitative spectraNucleus Experiment Quantitative Comments1H Single-pulse ✓ Recycle delay long enough (5-10s)

19F Single-pulse ✓ Recycle delay long enough (1-60s) Background interference

13C Single-pulse w/ 1H decoupling ✗ nOe enhances signals of sites with

hydrogens

Alternative is to use Inverse-gated decoupling Allow long relaxation time before each scan (10-60s)

31P Single-pulse w/ 1H decoupling ✗ nOe enhances signals of sites with

hydrogens

Alternative is to use Inverse-gated decoupling Allow long relaxation time before each scan (5-30s)

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Baseline Correction• Accurate intgration requires good definition of zero intensity (i.e.,

baseline) • Automatic Routines (polynomial or spline) work well for most small

distortions • In some cases may need other methods

Portion of signal below zero contributes a negative value to sum (integral)

After BC

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Baselines due to ‘stuff’ in probe• NMR probes are made of stuff • Borosilicate glass and quartz (10/11B and 29Si) • Fluoropolymers (19F and sometimes 13C) • Metals in the detection coil (63/65Cu, 195Pt)

• Result in obtrusive background signals/baselines

19F

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Broad signals, short FIDs

Solution is to ‘remove’ offending points Then, use backwards linear prediction

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Linear Prediction• Time-domain (FID) fitting routines to replace missing data • Existing points used as a basis set • Backwards linear prediction generates point at beginning • Useful if long delay before acquisition (alternative to very large ph1)

x

time

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19F

As-collected

Removal of initial points & Backwards Linear Prediction

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Are integrals Accurate?• Integrals in most software packages

done numerically • Works well when signal-to-noise is high

• Alternative is lineshape fitting • Works will even if signal-to-noise is low

Numerical Integration

Lineshape Fitting

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Real Example lineshape Fits: Pt-195 NMR

Samples Courtesy: Imelda Silalahi and Duncan Bruce, University of York

High signal-to-noise signal Integration and lineshape similar result

Low signal-to-noise signal Integration yields erratic result Lineshape fit yields consistent result

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Bandwidth and Integrals• Probe and Pulses utilised have intrinsic bandwidths • Peaks very far apart may be affected in different ways • Can be issue for 19F if very large shifts are present • E.g., Ar-F (130 ppm) vs. M-F (-350 ppm)

667 ppm

400 ppm

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Coupling to Quadrupolar Nuclei• Quadrupolar nuclei can often have T1s comparable to 1/J

[(R)2P)2Co-H

Magnetically equivalent phospines P-31 (N.A. 100%, I-1/2) Cobalt Co-59 (N..A. 100%, I-7/2)

Expect octet of triplets Observe a blob?

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1/J is a measure of time it takes a nucleus to ‘tell’ another nucleus what state its in.

If T1 comparable to 1/J, then the spin-state can change before a nucleus has time to ‘tell’ another nucleus what state it is in

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[(R)2P)2Co-H

Magnetically equivalent phospines P-31 (N.A. 100%, I-1/2) Cobalt Co-59 (N..A. 100%, I-7/2)

1H spectrum with 59Co decoupling results in triplet (due to 31P J-coupling)

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DEPT-135 vs 13C 1D

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Correlation Spectroscopy (i.e., 2D NMR)• Collect a series of 1D spectra where some period of additional

evolution occurs • Evolution of states during t1 (often linked via J-coupling)

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something something else1st

evo

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2nd evolution

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2nd evolution


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2nd evolution

something something else

2nd evolution1st evolution

…

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Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity

ppm

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Connectivity limited to 4 or 5 bonds

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Connectivity limited to 4 or 5 bonds

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Total Correlation Spectroscopy (TOCSY)• Provides ‘all’ correlations irrespective of magnitude • Allows distinction of coupling networks • Does not permit identification of ‘neighbouring’ spins

rd

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t1

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mix

• Homonuclear Hartman-Hahn (HOHAHA) mixing • Mixing times • 10-30 ms: 1-5 bonds • 40-100 ms: 1-10 bonds

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TOCSY vs COSY

COSY TOCSY

Correlations between individual coupled spins

Correlations between all spin in a spin-system

Limited to 4 bonds Beyond 4 bonds Correlations limited by presence of non-H bearing atoms

Follow coupled spins sequentially to understand connectivity

Allow identification of groups of spins from specific parts of molecule e.g., sugar rings in polysaccharide Sidechains in peptide/protein

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Spin or chemical Exchange : NOESY/EXSY• Nuclear Overhauser effect • Dipolar cross-relaxation • ‘COSY-type’ signals may also be present

rd

90

t2

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t1

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mix

• No RF applied during mixing • Mixing times • 50-800 ms

• Negative correlations = nOe • Positive correlations = Xchng

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Distance Information from nOe• nOe inensity is related to the distance between the two nuclei

• Acquisition of spectra at different mixing times can allow extraction of distances

Figure from: P. Brocca, P. Berthault, S. Sonnino; Biophysical Journal (1998), 74(1), 309

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When NOESY doesn’t work…• Nuclear Overhauser effect near zero for molecules of MWt.

800-2000 (depending on solvent)

rd t2

90

t1 mix

• Use rOe (rotating frame Overhauser effect)

• Mixing times • 5-100 ms

• 2D spectrum similar to NOESY

• TOCSY-like peaks may be present

Small molecules

large molecules

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Heteronuclear Correlation• Used for probing connectivity between two different types of

nuclei

rd1/2J

t1

1H

X

1/2Jt2

rd1/4J

t1

X

t2

1H

1/4J 1/4J 1/4J

HMQC

HSQC

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Some Common H-X J-coupling ranges• 13C • One-bond: 120-170 Hz • Two-bond: <30 • Three-bond: <15

• 31P • One-bond: 30-1000 • Two-bond: <50 • Three-bond: <40

• 15N • One-bond: 65-100 Hz

• Two-bond: < 11Hz • Three-bond: < 11 Hz

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HSQC vs HMQC• HSQC • Heteronuclear Single-Quantum Correlation • 1H-detected 1H-X correlation spectrum

• Typically used to probe ‘one-bond’ correlations • Can be higher resolution

• Variants with multiplicity (XH, XH2, XH3 etc.) selection

• HMQC • Heteronuclear Multiple-Quantum Correlation

• 1H-detected 1H-X correlation spectrum

• Typically used to probe ‘one-bond’ correlations • can be higher sensitivity

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HSQC vs. HMQC

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• HSQC capable of intrinsically higher resolution than HMQC

1H{13C} HSQC 1H{13C} HMQC

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Edited HSQC• Adds a ‘DEPT-like’ selection • XH and XH3 positive

• XH2 negative

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Weak signals in HSQC• Weak signals in HSQC can arise from various sources • Minor impurities/byproducts • Multiple-bond correlations

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Multiple-Bond Correlation

• Heteronuclear Multiple-bond correlation experiment

• Multiple-bond correlations have smaller J-couplings

• HMBC effectively an HMQC with 1/2J set to optimize for smaller J-coupling • Often have an additional evolution to minimize signals from correlations

between sites with large J-couplings

• Use together with HSQC/HMQC to determine C-C connectivity

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HMQC/HSQC plus HMBC• Use HMQC/HSQC together with HMBC to determine

connectivity

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connectivity

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Single-bond correlation

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connectivity

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connectivity

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connectivity

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connectivity

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?

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NO Forward Linear Prediction

With Forward Linear Prediction

Linear Prediction: indirect dimension17 min

17 min

4 min

4 min

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Relevant LiteratureBooks • “Modern NM Techniques For Chemistry Research”, A.E. Derome • “High-Resolution NMR Techniques in Organic Chemistry”,

T.D.W. Claridge • “Nuclear Magnetic Resonance” , P.J. Hore (Oxford Primer) • “Spin Dynamics”, M.H. Levitt

Journals/Book Series • Concepts in Magnetic Resonance • Annual Reports in NMR (Chemistry Library) • Encyclopedia of NMR (DH Coffee room)

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NMR: PRACTICAL ASPECTS

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