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NMR at UNC: Tips, Tricks, and Techniques
Laura Adduci
UNC-Chapel HillGraduate student – Gagné Lab
NMR Assistant – Chemistry Department
Topics and Examples
1. 2D spectra
2. Selective 1D spectra
3. Homonuclear and heteronuclear decoupling experiments
4. Spectrometers at UNC
O OMe
OHHOOH
HO
O OH
OHHOOH
HO
HOOH
OHO
HO
O
OHOH
HO
HOO
Compounds in example spectra:
O OH
OHHOOH
HO
Standard 1D spectrum
Each resonance has:1. Chemical shift2. Integration3. Multiplicity/coupling
constant (e.g. 3JH,H = 6 Hz)4. Phase
Negative phasing:
2D COSY and TOCSY
COSY
COSY
TOCSY
TOCSY
12 13
14,1511
7 12 13
14,1511
7
7
12
13
11
14,15
7
12
13
11
14,15
Default: 5 minutes Default: 75 minutes
COSY: Correlates coupled protons (usually 2JH,H and 3JH,H) TOCSY: Correlates protons in the same spin system
2D NOESY
NOESY: Correlates protons that are near each other in space
46
13
7
1213
1114, 15
4, 61, 3
7
12
1311
14, 15 Default: 45 minutes
2D HSQC and phase-sensitive HSQC
HSQC
HSQC
HMQC
HMQC
HSQC
HSQC
14,15
1213
11 14,15
13
1114,15
13
11
1213
14,1511
HSQC: Correlates protons and the carbons to which they are directly bound (1JC,H)
Phase-edited: CH3 and CH peaks appear as one phase, CH2 peaks appear as different phase
Default: 15 minutes Default: 15 minutes
12,7 12,7
O
OHCH2
1
23
4
5
6 78
1213
15
14
11
2D HMBC
O
OH
1
234
5
6 78
1213
15
14
11
H
13 4
67
1213
1114, 15
8
51,3
24,6
7,12
1314,15 11
HMBC: Correlates protons with carbons that are 2 and 3 bonds away (2JC,H and 3JC,H)
Default: 15 minutes
1JC-H coupling: appears as weak doublets, usually visible for
sharp/strong proton peaks only
Choosing parameters for 2D experiments
Parameters that can be varied:1. Number of scans (type “ns”): affects signal-to-noise ratio
Increase if you have low sample concentration
2. Time domain in F1 (type “td”): affects resolution in the F1 dimension (vertical)Increase if your peaks are close together
3. Experiment-specific parametersTOCSY: TOCSY mixing time (d9)
• Affects the number of bonds through which magnetization is transferred• Increase to see correlation between distant protons in the same spin
system• Typical values: 10 ms – 400 ms
NOESY: NOESY mixing time (d8)• Affects NOE buildup between correlated nuclei• Typical values: 10 ms – 900 ms
TD(F1)
NS
Alternative to 2Ds: selective 1D experiments
1. Selectively irradiate one peak in a 1D spectrum
2. Apply pulse sequence analogous to a certain 2D experiment
3. Only nuclei that are correlated to the irradiated peak by the applied method are observed in the new 1D spectrum
Example: 1. Irradiate here
2. Apply COSY-like pulse sequence
3. Expect to see peak for this proton in addition to the
initially irradiated proton
Selective 1D COSY
O OMe
OHHOOH
HO 12
345
6,6'1
23 5 4
Me
6 6’
1
2
23
4
1
6’65
4
66’
5
Default: 1 minute
Choosing selective 1D’s vs 2D
Selective 1D’s are beneficial if:1. Limited sample concentration2. Overlapping peaks3. You want to see “shape” of correlated peaks, not just whether or not there is a correlation4. Many correlations expected, but only a few are of interest
Selective 1D NOESY example
Mike Geier
Default: 3 minutes
Selective 1D TOCSY of sucrose
HOOH
OHO
HO
O
OHOH
HO
HOO
G
F
GG
GGF F
F
G, F
G
G
G
G
GG
F
F
F F
G
Default: 1 minute
Separation of peaks from two different spin systems
Selective 1D TOCSY with two products
Trandon Bender
Z E
Z
E
Z
E
Separation of peaks from two different compounds in sample
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: step by step
Selective 1D TOCSY: increasing mixing time
TOCSY mixing time
d9 = 120 ms
d9 = 60 ms
d9 = 40 ms
d9 = 20 ms
d9 = 10 ms
2
3
5
4
6’6
1
Mixing time affects the number of bonds through which magnetization is transferred
Homonuclear decoupling
16
235 4 123456
Selectively irradiate at one frequency. Coupling to the peak at that frequency is not observed in other peaks.
23
1Example: irradiate at H2. Coupling to H2 is
suppressed from H1 (triplet becomes singlet) and H3
65
45 3
Should never conflict with COSY data!
Heteronuclear decoupling: 1H{31P}
31P decoupling
Spectrometers at UNC
Remember: signal-to-noise increases as the square of the number of scansexample: to multiply signal-to-noise by 2, must multiply number of scans by 4
299259 383 17301H
824843605 525613C
400 NB 400 WB 500 (BBO) 600 (CryoQNP)
“Drop‐off NMR” on the Bruker 600: Samplechanger + spreadsheet
Signal‐to‐noise ratios
Tuesdays & Fridays at 12:00 pm Thursdays at 6:00 pm (overnight)
Holder # Onyen Data set name Solvent Experiment Title Advisor Grant number Mail Data/Notify to: Directory11 bezier DB834ALLEXP C6D6 Proton_C13‐512_COSY_HMQC_HMBC_ DB834ALLEXP Brookhart 544705 [email protected] C:\data\bezier12 samander SA2_151_pure CDCl3 Proton_C13‐512 SA2_151_pure PADI PO You 535778 [email protected] C:\data\samand13 dabrowje JAD‐I‐067‐2F7‐14 CDCl3 C13‐256 JAD‐I‐067‐2F7‐14 Gagne 535902 [email protected]:\data\dabrow14 bvass BFV 27 [Ru(b)(phen)(COCD2Cl2 PROTON BFV 27 [Ru(b)(phen)(C Schauer 535944 [email protected] C:\data\bvass17 rrwatkin RW‐1‐12 p‐vinylphenylaDMSO PROTON UAA after deprotectio Brustad 555083 [email protected] C:\data\rrwatk18 wilger DW_02_053_A CDCl3 PROTON DW_02_053_A Nicewicz 545715 [email protected] C:\data\wilger19 wilger DW_02_053_B CDCl3 PROTON DW_02_053_B Nicewicz 545715 [email protected] C:\data\wilger20 wilger DW_02_053_C CDCl3 PROTON DW_02_053_C Nicewicz 545715 [email protected] C:\data\wilger21 wilger DW_02_053_D CDCl3 PROTON DW_02_053_D Nicewicz 545715 [email protected] C:\data\wilger22 wilger DW_02_053_E CDCl3 PROTON DW_02_053_E Nicewicz 545715 [email protected] C:\data\wilger23 tbender TAB2‐76‐A‐DMSO DMSO Proton_C13‐512_COSY_HSQC TAB2‐76‐A‐DMSO Gagne 535901 [email protected]:\data\tbende
394
Varian 600
Summary
• 2D experiments help with compound characterization
• 1D selective experiments complement 2D experiments and sometimes provide more
information
• 1D selective TOCSY spectra can be used to separate peaks from two different species
in solution
• Selective decoupling can help elucidate coupling constants
Thanks to…
• Ben Giglio• Dr. Marc ter Horst
• Mike Geier• Trandon Bender
Extra slides
Heteronuclear decoupling: 1H{19F}
Ben Giglio
-OH-CH2-Standard 1H spectrum
1H spectrum with 19F decoupling
Irradiate one nucleus – coupling to that nucleus is suppressed.Most common: proton-decoupled carbon spectrumCan use this method for other nuclei as well.
Horizontal axis is proton spectrum.Vertical axis is “peak separation” in Hz
J-resolved spectrum
1 Hz
HOOH
OH
OH
OH
OH1233'2'1'
2
132
13
6.5 Hz
3JH,H = 6.5 Hz
13C APT and DEPT spectra
A CB
FEG
D
A B
CD E
FG
13C{1H}: all peaks positive
13C APT: carbons with 1 or 3 attached protons are negative, carbons with 0 or 2 protons are positive (and a deuterium is not a proton!)
DEPT 135: carbons with 1 or 3 protons are positive, carbons with 2 protons are negative, carbons with 0 protons are attenuated
DEPT 90: carbons with 1 proton are positive, all others are attenuated
“Quantification” of 13C NMR
OA
BCD
E FG
128 scans Regular 1H decoupling Inverse gated decoupling
Without Cr(acac)3
d1(s) 0.3 0.5 1 2 10 20 0.3 0.5 1 2 10 20time 2:47 3:14 4:20 6:32 24:08 46:08 2:43 3:10 4:16 6:28 24:04 46:04Int A 0.60 0.71 0.84 1.00 1.50 1.62 0.62 0.68 0.79 0.94 1.25 1.32Int B 0.74 0.83 0.97 1.10 1.62 1.78 0.71 0.79 0.91 1.08 1.30 1.30Int C 2.16 2.32 2.53 2.84 3.43 3.54 2.08 2.18 2.37 2.47 1.91 1.52Int D 2.70 2.95 3.14 3.38 3.73 3.81 2.75 2.84 2.90 2.77 1.64 1.36Int E 2.76 2.92 3.10 3.34 3.72 3.78 2.79 2.86 2.94 2.79 1.63 1.37Int F 2.40 2.48 2.72 3.01 3.56 3.64 2.36 2.43 2.60 2.63 1.78 1.45
Int G 2.80 2.84 3.10 3.36 3.78 3.83 2.71 2.74 2.82 2.69 1.59 1.36
With Cr(acac)3
d1(s) 0.3 0.5 1 2 10 20 0.3 0.5 1 2 10 20time 2:47 3:14 4:20 6:32 24:08 46:08 2:43 3:10 4:16 6:28 24:04 46:04Int A 0.99 0.98 1.00 1.00 1.01 1.00 0.98 0.96 1.00 0.99 0.98 0.99Int B 0.95 0.97 0.98 0.96 0.99 0.98 0.95 0.96 0.99 0.99 0.96 0.98Int C 1.12 1.14 1.12 1.14 1.15 1.15 1.05 1.05 1.02 0.99 1.01 1.01Int D 1.19 1.20 1.23 1.23 1.23 1.25 1.11 1.07 1.01 0.99 0.98 0.96Int E 1.21 1.22 1.21 1.23 1.23 1.23 1.12 1.09 1.02 0.99 0.98 0.99Int F 1.14 1.15 1.17 1.16 1.15 1.15 1.08 1.05 1.04 1.01 1.01 1.01
Int G 1.22 1.22 1.22 1.21 1.23 1.23 1.12 1.09 1.04 0.99 0.98 0.99
What factors affect peak integration?1. Number of attached protons: NOE during decoupling gives NOE enhancement
2. Relaxation delay: peaks with T1’s that are longer than the relaxation delay are attenuated
Solution: can use inverse gated decoupling. Decoupler is on during acquisition so that peaks appear decoupled but off during delay so that there is no time for the NOE to build up
Solution: add relaxation agent to shorten all T1’s, or lengthen relaxation delay
Overlapping Kinetics Using the Samplechanger
• Staggered kinetics runs• Start one run, then a second (third, fourth…)
during the delay between timepoints• Allows multiple kinetics runs to be acquired in
the same amount of time as one run• Minimum time between samples:
5 minutes + acquisition time • Works best when samples are similar (same
solvent)Sample 1
Sample 1
Sample 2
12
6
39
10 2
8 47 5
11 1
Sample 3
Sample 3
Sample 2
30 min
30 min
30 min
30 min
30 min
