how to analyze of 2d nmr spectra ( small molecules) 2009. 12. 28 노 정 래
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How to Analyze of 2D NMR Spectra ( small molecules)
2009. 12. 28
노 정 래
Pulse width(pw)
FT
1H
Conventional proton spectrum (1H NMR)
ppm2345678
ppm405060708090100110120130140150160170
1H and 13C NMR spectra (Chemical shifts, )
H
HO
H
NH
O
H
C
C
COCN
C
O
Carbon spectrum (13C NMR)
FT
1H 13C
CHCl3 (1% ) 13C1H 13C1H
13C
Bo
(Coupled carbon spectrum)
couplingJCH
Pulse width(pw)
13C
BB 13C1H
Carbon spectrum (13C NMR)(Decoupled carbon spectrum)
1H 13C
CHCl3 (1% )
13CH313CH2
13CH 13C
C C C C
H
H
H
H
H
H
Coupled Carbon
Decoupled Carbon Nuclear Overhauser Effect (nOe)
Proton-Proton coupling constant (JHH)
H
H
JHHJHH
JHH
Not through space, but through bonds
- Hybridization of the atoms- Bond lengths- Bond angles and dihedral angles- Substituent effects- The presence of neighboring -bonds
JHH [Hz] sign JCH [Hz] sign JCC [Hz] sign
1J 125 ~ 250 + 30 ~ 80 +
2J 0 ~ 30 -* -10 ~ 20 + / - < 20 + / -
3J 0 ~ 18 + 1 ~ 10 + 0 ~ 5 +
3+nJ 0 ~ 7 + / - < 1 + / - < 1 + / -
* Usually negative, but sometimes positive
Factors influencing scalar coupling
The order of magnitude and sign of scalar couplings
2-methylpent-1-en-3-ol
Connectivity of protons and carbons
OH
Men and their Partner (Direct Coupling)
Friendship of Partners (Indirect coupling)
Relation between Men and their friend’s partner (remote coupling)
• Direct Detection
New Techniques employed in modern NMR experiments
low sensitivity (low natural abundance)
long time for multidimensional NMR experiment
high sensitivity
short time for multidimensional NMR experiment
13C1H
• Indirect Detection (Inverse Detection)
13C1H
99%( inactive)
1% (active)
12C1H
13C1H
labeling(70% 13C)
natural (1% 13C)
H-C-Cl3
JCH=216 Hz
+ =
Comparison of spectrum using phase cycling and PFG
• Phase cycling ( it is necessary to select interested signals with several scans)
scan 1 scan 2 sum
• PFG ( select interested signals or eliminate the unwanted signals with one PFG pulse
One scan
In case of dense sample - gives spectrum in short time - gives clean spectrum
PFG
dz
Field strength
1H
x
y
Bo
x
y
Pulse Field Gradient (PFG)
PFG
x
y
x
y
x
y
Pulse Field Gradient (PFG)
Origin 2D Data
t J
t2
t1
t1
0 200 400 600 800 1000t2 pts
0.1220
0.1230
0.1240
0.1250
0.1260
0.1270
t1 sec
Production of 2D NMR spectrum
FT (t2)
t2t1
FT (t1)
t2
t1
1H
1H
F1
F2
Ha
Hb
HaHb
cross peak diagonal peaks
Ha / Hb
COSY spectrum
t1 t2
128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)512 x 1024 (1024 x 2048)
90 90
COSY spectrum (COrrelation SpectroscopY)
C
Hb
C
Ha
2JHH
C Hb
Ha
3JHH
Ha Hb
4JHH1
23
4
t1
t2
= 0-30 Hz
= 0-18 Hz
FT(t1, t2)
C C C C C C
Ha Hb HcHd He
a b c ed
a
b
d
c
e
diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak
COSY spectrum and its Interpretation
a
b
d
c
e
Hb / Hc
C C C C C C
Ha Hb HcHd Hf
a bc fd
a
b
e
c
f
( )
diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak
He
COSY spectrum and its Interpretation
e
d
C C C C C C
Ha Hb HcHd He
a b/ec fd
a
b/e
d
c
f
( )
diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak
C
Hf
Overlapped signals
COSY spectrum and its Interpretation
b c
b d
TOCSY (TOtal Correlation SpectroscopY)
d b c a
a
c
b
d
t1t2
128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)512 x 1024 (1024 x 2048)
90 Mixing pulse60~80ms
C C C C
Ha Hb HcHd
3JHH3JHH
3JHH1
2
3
t1 t2
4
FT(t1, t2)
TOCSY spectrum
C C C C C C
Ha Hb HcHd He
a b/ec fd
a
b/e
d
c
f
C
Hf
d
a
c
b/e
f
TOCSY spectrum and its Interpretation
13C
CaCb
Ha
Hb
1H
Ca
Cb
13C
1H
HaHb
HMQC (Heteronuclear Multiple Quantum Correlation) / HSQC (Heteronuclear Single Quantum Correlation)
t1
t2
1/2JCH
128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)
Pairing 1H and 13C Shifts by using the HSQC / HMQC spectrum
= 125-250 Hz1JCH
13C 1H
Ca Cb O
1Ha 1Hb
1
3
t1 t24
1JCH2
1JCH1JCH
FT(t1, t2)
HSQC spectrum
C C O C C
OH
H
H H H
H H
H
HMQC/HSQC spectrum and its Interpretation
abc d
a b c d
CH3, a
CH3, d
CH2, b
13C
1H
HMBC (Heteronuclear Multiple Bond Correlation)
t1
t2
128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)
90 1801/2JCH
1/2nJCH
Assignment of Nonprotonated 13C’s on the basis of the HMBC spectrum
FT(t1, t2)
HMBC spectrum
Ca Cb O Cc Cd
OHa
Ha
Ha Hb Hd
Hb Hd
Hd
adbc
a
d
b
HMBC spectrum and its Interpretation
1H
13C
diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak
NOESY/ROESY( Nuclear Overhauser Effect Spectroscopy)
t1 t2
90 9090
t1t2
90 Mixing pulse350 ms
Mixing pulse200 ms
Determining Stereochemistry by using the NOESY / ROESY spectrum
C C C
HaHb Hc
NOE
t1
t2
NOE
H H
H
a b
c
2JHH
NOE / COSY
NOE
NOESY
ROESY
FT(t1, t2)
NOESY/ROESY spectrum
DEPT (Distortionless Enhancement by Polarization Transfer)
CH
CH2
CH3
1/2J1/2J 1/2J
x90
x180
x90
y180
y
H3C C
O
CH
CH3
CH2OH
220 200 180 160 140 120 100 80 60 40 20 0220 200 180 160 140 120 100 80 60 40 20 0
a
bcd
e
a
b c d e
13C spectrum
DEPT-135(CH + CH3 - CH2)
DEPT- 90 (CH)
DEPT- 45 (CH + CH2 + CH3)
DEPT spectra
4-hydroxy-3-methyl-2-butanone
13C spectrum
DEPT- 45
DEPT- 90
(DEPT- 45)-(DEPPT-135)
(DEPT- 45)+(DEPT-135)-(DEPT- 90) CH3
CH2
CH
All protonated
Pure subspectra
DEPT spectrum and its interpretation
I . Assignment of Resonances to Atoms Within a MoleculeI . Assignment of Resonances to Atoms Within a Molecule
Assigning 1H Resonances on the basis of Chemical Shifts
Assigning 1H Resonances on the basis of the COSY spectrum
Assigning 13C Resonances on the basis of Chemical Shifts
Pairing 1H and 13C Shifts by using the HSQC / HMQC spectrum
Assignment of Nonprotonated 13C’s on the basis of the HMBC spectrum
Determining Stereochemistry by using the NOESY / ROESY spectrum
II. Elucidation of Unknown Molecular StructuresII. Elucidation of Unknown Molecular Structures
Initial Inspection of the one-dimensional spectra : 1H and 13C
Establishment of connectivity between protons on the basis of the gCOSY spectrum
List the1H -13C data in tabular form
Pairing 1H and 13C Shifts by using the HSQC / HMQC spectrum
Assignment of Nonprotonated 13C’s on the basis of the HMBC spectrum
Determining Stereochemistry by using the NOESY / ROESY spectrum
C9H9ClO2 MW=184.62
* Problem 1 and 2 are selected from “Organic Structure Determination Using 2-D NMR Spectroscopy” - J. H. Simpson, 2008
Problem 1
1H NMR
The carbon signal at 157.0 ppm is lost
13C NMR
HMQC
1H COSY
NOESY
C7H12O2
Problem 2
1H NMR
13C NMR
HSQC
1H COSY
HMBC
10.88.06
(d, 7.7)
7.37(d, 8.0)
7.24(dd, 8.0, 7.4)
7.04(dd, 7.7, 7.4)
3.64
2.33 2.26
Problem 3
1H spectrum
x x x
13C spectrum
70%M-hsqc.esp
120 110 100 90 80 70 60 50 40 30 20F2 Chemical Shift (ppm)
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
F1
Ch
em
ica
l S
hift (p
pm
)
HSQC
70%M-cosy.esp
8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9F2 Chemical Shift (ppm)
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2
F1
Che
mic
al S
hift
(pp
m)
COSY Spectrum8.06 7.37 7.24 7.04
70%M-hmbc.esp
150 145 140 135 130 125 120 115 110 105 100 95F2 Chemical Shift (ppm)
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
F1
Ch
em
ica
l S
hift (p
pm
)
HMBC Spectrum
70%M-hmbc.esp
145 140 135 130 125 120 115 110 105F2 Chemical Shift (ppm)
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
F1 C
hem
ical S
hift (ppm
)
Expanded HMBC Spectrum
12
7.0
10
8.5
13
8.3
13
6.9
70%M-hmbc.esp
140 135 130 125 120 115 110F2 Chemical Shift (ppm)
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2
F1 C
hem
ical S
hift (ppm
)
Expanded HMBC Spectrum
13
9.5
12
3.8
12
2.8
12
1.9
11
8.0
11
0.2
70%M-hmbc.esp
140 135 130 125 120 115 110F2 Chemical Shift (ppm)
10.75
10.76
10.77
10.78
10.79
10.80
10.81
10.82
10.83
10.84
10.85
10.86
10.87
F1 C
hem
ical S
hift (ppm
)
Expanded HMBC Spectrum
10
9.6
12
2.8
13
6.9
13
9.5
문제 풀이
C9H9ClO2 MW=184.62
Problem 1
1H NMR
Degrees of unsaturation = 9 – 9/2 + 1-1/2 =10
7.22
6.85
4.20 3.903.34
2.90 2.75
* The carbon signal at 157.0 ppm is lost
13C NMR
HMQC 129.4 115.9
69.0 50.0 44.6
7.22
6.85
4.20
3.90
3.34
2.90
2.75
129.4 - 7.22115.9 – 6.85 69.0 – 3.90 / 4.20 50.0 – 3.34 44.6 – 2.75 / 2.90
1H COSY
XH
HH
H
129.4 - 7.22115.9 – 6.85 69.0 – 3.90 / 4.20 50.0 – 3.34 44.6 – 2.75 / 2.90
7.22
6.85
3.904.20
3.34
2.752.90
O
NOESY
H
HH
H 7.22
6.853.904.20
2.752.90
O3.34
O
C9H9ClO2
O
Cl
O
Cl
O
C9H9ClO2
C9H9ClO2
C7H12O2
Problem 2
1H NMR
6.15
4.90
4.683.79 3.50
2.07 1.86
1.76
1.16Degrees of unsaturation = 7 – 12/2 + 1 =2
13C NMR
HSQC
140.4 101.1 63.5
26.8
16.3
15.0
96.7
6.15
4.90
4.68
3.793.50
2.071.86
1.76
1.16
140.4 – 6.15101.1 – 4.68 96.7 – 4.90 63.5 – 3.50 / 3.79 26.8 – 1.76 (CH2) 16.3 – 1.86 /2.07 15.0 – 1.16 (CH3)
H H
1H COSY
6.15
4.90
4.68
3.79
3.50
1.861.76
1.16
2.07
140.4 – 6.15101.1 – 4.68 96.7 – 4.90 63.5 – 3.50 / 3.79 26.8 – 1.76 (CH2) 16.3 – 1.86 / 2.07 15.0 – 1.16 (CH3)
6.15 4.68
1.862.07
1.76
4.90 O1.16
3.503.79
HMBC
C7H12O2 H H6.15 4.68
1.862.07
1.76
4.90 O1.16
3.503.79
140.4 – 6.15101.1 – 4.68 96.7 – 4.90 63.5 – 3.50 / 3.79 26.8 – 1.76 (CH2) 16.3 – 1.86 / 2.07 15.0 – 1.16 (CH3)
OH
H
O
Degrees of unsaturation = 2
10.8
8.06(d, 7.7)
7.37(d, 8.0)
7.24(dd, 8.0, 7.4)
7.04(dd, 7.7, 7.4)
3.64
2.33 2.26
Problem
1H spectrum
MW = 241C15H15NO2
Degrees of unsaturation = 15 -15/2 + 1 +1/2 = 9
x x x
13C spectrum
70%M-hsqc.esp
120 110 100 90 80 70 60 50 40 30 20F2 Chemical Shift (ppm)
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
F1
Ch
em
ica
l S
hift (p
pm
)
HSQC Spectrum
142.6
139.5
138.3
136.9
127.0
123.8 - 7.24
122.8
121.9 - 8.06
118.0 - 7.04110.2 - 7.37109.6108.5 60.6 - 3.64 (CH3) 13.4 - 2.33 (CH3) 12.5 - 2.26 (CH3)
70%M-cosy.esp
8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9F2 Chemical Shift (ppm)
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2F
1 C
hem
ical
Shi
ft (p
pm
)
COSY Spectrum
H
H
H
H
8.06 7.37 7.24 7.04
8.06
7.04
7.24
7.37
70%M-hmbc.esp
150 145 140 135 130 125 120 115 110 105 100 95F2 Chemical Shift (ppm)
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
F1
Ch
em
ica
l S
hift (p
pm
)
HMBC Spectrum
70%M-hmbc.esp
145 140 135 130 125 120 115 110 105F2 Chemical Shift (ppm)
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
F1 C
hem
ical S
hift (ppm
)
X
CH3
O
CH3
CH3
Expanded HMBC Spectrum
12
7.0
10
8.5
13
8.3
13
6.9
138.3
108.5
127.0
136.9
142.6
139.5
138.3
136.9
127.0
123.8 - 7.24
122.8
121.9 - 8.06
118.0 - 7.04110.2 - 7.37109.6108.5 60.6 - 3.64 (CH3) 13.4 - 2.33 (CH3) 12.5 - 2.26 (CH3)
70%M-hmbc.esp
140 135 130 125 120 115 110F2 Chemical Shift (ppm)
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2
F1 C
hem
ical S
hift (ppm
)
H
H
H
H
Expanded HMBC Spectrum
8.06 / 121.9
7.04 /118.0
7.24 / 123.8
7.37 / 110.2
139.5
122.8
13
9.5
12
3.8
12
2.8
12
1.9
11
8.0
11
0.2
8.06
7.37
7.24
7.04
70%M-hmbc.esp
140 135 130 125 120 115 110F2 Chemical Shift (ppm)
10.75
10.76
10.77
10.78
10.79
10.80
10.81
10.82
10.83
10.84
10.85
10.86
10.87
F1 C
hem
ical S
hift (ppm
)
Expanded HMBC Spectrum
10
9.6
12
2.8
13
6.9
13
9.5
H
H
H
H
139.5
122.8
X
CH3
O
CH3
CH3
136.9
109.5
NH
10.2
10.2
C15H15NO2
Degrees of unsaturation = 9
NH
OH
OCH3MW = 241C15H15NO2