NMR: PRACTICAL ASPECTS Pedro M. Aguiar 1 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) • 1 H NMR: >= 10 mM • 13 C 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 2
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NMR: PRACTICAL ASPECTSPedro M. Aguiar
1
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
2
Sample PreparationAssumed detection region for shimming
ideal
reality
Good 35-45 mm
3-1
Sample PreparationAssumed detection region for shimming
ideal
reality
• Poor shimming • Weak signal (not in
detector)
Short < 35 mm
Good 35-45 mm
3-2
Sample PreparationAssumed detection region for shimming
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
3-3
Sample Volume: 5 mm NMR tube
600 µL volume Well-shimmed Good lineshape
300 µL volume poorly-shimmed Poor lineshape
4
Limited Sample volume• In cases of Limited sample volume partially filled 5 mm tube
Exponential Apodisation (line broadening) is most common for 1D
Optimum is to use “LB” of 0.5-1 times FWHM of peaks
€
e−πLBt
8
Apodisation and Processing
FT
9
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
2.0
2.0
10
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
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)
12
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
13
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
14
Broad signals, short FIDs
Solution is to ‘remove’ offending points Then, use backwards linear prediction
15
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
16
19F
As-collected
Removal of initial points & Backwards Linear Prediction
17
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
18
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
19
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
20
Coupling to Quadrupolar Nuclei• Quadrupolar nuclei can often have T1s comparable to 1/J
1H spectrum with 59Co decoupling results in triplet (due to 31P J-coupling)
23
DEPT-135 vs 13C 1D
24
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)
25-1
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)
something something else1st
evo
l.
2nd evolution
25-2
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)
something something else1st
evo
l.
2nd evolution
something something else1st
evo
l.
2nd evolution
25-3
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)
something something else1st
evo
l.
2nd evolution
something something else1st
evo
l.
2nd evolution
something something else
2nd evolution1st evolution
…
25-4
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
26
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
Connectivity limited to 4 or 5 bonds
27-1
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
Connectivity limited to 4 or 5 bonds
27-2
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
28-1
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
28-2
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
28-3
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
29-1
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
29-2
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
29-3
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
29-4
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
30-1
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
30-2
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
30-3
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
30-4
Correlation Spectroscopy (i.e., 2D NMR)• COrrelation SpectroscopY (COSY) • One of most basic 2D experiments • Information about connectivity
ppm
5 4 3 2 1 0 ppm
5
4
3
2
1
0
rd
90
t2
90
t1
30-5
Total Correlation Spectroscopy (TOCSY)• Provides ‘all’ correlations irrespective of magnitude • Allows distinction of coupling networks • Does not permit identification of ‘neighbouring’ spins