NMR spectrum Structure Chem. Shift J Coupling Peak Int. NOE Correlation H-H ， C-H Applications: Sample quality control for Synthetic works. Elucidation.

NMR spectrum

Structure

Chem. Shift

J Coupling

Peak Int.

NOE

CorrelationH-H ， C-H

O

OOH

O

CH3

O

H

OH

OH

OH

CH2OH

COOCH2

1

2

3

4

5

6

7

8

9

10

1''

2''3''

4''

5'' 6''

1'

2'3'

4'

5'

6'

7''

Applications:• Sample quality control for Synthetic works.• Elucidation of chemical structures. • Getting functional group, bonding, dynamics, kinetics and

chemical exchange information of molecules.• 3D structures of the molecules.

NMR Applications in Chemistry

Connections through spaceConnections through space （（ dipolar couplingdipolar coupling ）） 1D1D 、、 2D NOESY2D NOESY ，， ROESY, HOESY(HSQC-NOESY)ROESY, HOESY(HSQC-NOESY)

usageusage ：： connecting spin systems connecting spin systems 、、 structure determinatistructure determinati

onon

Connections through bondsConnections through bonds （（ spin-spin couplingspin-spin coupling ）） HomonuclearHomonuclear ：： 1D1D 、、 2D 2D 11H-H-11H COSY, DQF-COSY, TOCSYH COSY, DQF-COSY, TOCSY

usageusage ：： spin system assignmentspin system assignment

HeteronuclearHeteronuclear ：： Direct (detect Direct (detect 1313C): C): APT, DEPT, HETCORAPT, DEPT, HETCOR

Inverse (detectInverse (detect11H): H): HMQC, HSQC, HMBC, HSQC-COSY, HMQC, HSQC, HMBC, HSQC-COSY,

HSQC-TOCSY, HMQC-TOCSYHSQC-TOCSY, HMQC-TOCSY

usageusage ：： assigning heteronucleiassigning heteronuclei 、、 connecting spin systemsconnecting spin systems

Some common NMR experiments:Some common NMR experiments:

COSY spectrum is used for determining the connectivitiesbetween protons on the basis of geminal and vicinal couplings.Disadvantage: bulky dispersive diagonal peaks.

C C H

HH

VicinalGeminal

2D COSY2D COSY — Homonuclear Shift — Homonuclear Shift COCOrrelation rrelation SSpectroscoppectroscopYY

t1 AQ

The basic COSY (x=45° or 90°) pulse sequence

90° x°t2

ppm

1.01.52.02.53.03.5 ppm

1.0

1.5

2.0

2.5

3.0

3.5

Current Data ParametersNAME butcosyEXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20001102Time 8.04INSTRUM DRX500PROBHD 5 mm TBI 1H/PULPROG cosygpTD 1024SOLVENT CDCl3NS 1DS 16SWH 2185.315 HzFIDRES 2.134096 HzAQ 0.2345700 secRG 40.3DW 228.800 usecDE 6.00 usecTE 288.0 KD0 0.00000300 secD1 1.60000002 secD13 0.00000300 secD16 0.00010000 secIN0 0.00045765 sec

============ CHANNEL f1 =============NUC1 1HP0 3.00 usecP1 6.00 usecPL1 -4.00 dBSFO1 500.1310815 MHz

============ GRADIENT CHANNEL ========GPNAM1 sine.100GPNAM2 sine.100GPX1 0.00 %GPX2 0.00 %GPY1 0.00 %GPY2 0.00 %GPZ1 10.00 %GPZ2 10.00 %P16 1000.00 usec

F1 - Acquisition parametersND0 1TD 256SFO1 500.1311 MHzFIDRES 8.535453 HzSW 4.369 ppmFnMODE undefined

F2 - Processing parametersSI 2048SF 500.1300144 MHzWDW QSINESSB 0LB 0.00 HzGB 0PC 0.20

F1 - Processing parametersSI 1024MC2 QFSF 500.1300144 MHzWDW QSINESSB 0LB 0.00 HzGB 0

11 22 33 4411

2233

44

HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

1-21-22-32-3

3-43-4

2D Gradient COSY-452D Gradient COSY-45

2D 2D DQFDQF －－ COSYCOSY((DDouble-ouble-QQuantum uantum FFiltered iltered COSYCOSY))

90 90 90

t1 t2

allow J coupling constant measurements allow J coupling constant measurements

observing cross-peaks close to diagonalobserving cross-peaks close to diagonal

But lower sensitivity (~40%) of COSY expt.But lower sensitivity (~40%) of COSY expt.

ppm

3.63.84.04.24.44.64.85.05.25.45.65.86.06.2 ppm

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

Current Data ParametersNAME gbc35dqfEXPNO 2PROCNO 1

F2 - Acquisition ParametersDate_ 20001103Time 4.01INSTRUM DRX500PROBHD 5 mm TBI 1H/PULPROG cosydfetgp.2TD 4096SOLVENT PyrNS 8DS 16SWH 3822.630 HzFIDRES 0.933259 HzAQ 0.5359376 secRG 512DW 130.800 usecDE 6.00 usecTE 288.0 KD0 0.00000300 secD1 1.60000002 secD11 0.03000000 secD16 0.00010000 secDELTA 0.00110300 secDELTA1 0.00110800 secIN0 0.00026161 secl3 256


============ GRADIENT CHANNEL ========GPNAM1 sine.100GPNAM2 sine.100GPNAM3 sine.100GPX1 0.00 %GPX2 0.00 %GPX3 0.00 %GPY1 0.00 %GPY2 0.00 %GPY3 0.00 %GPZ1 30.00 %GPZ2 10.00 %GPZ3 50.00 %P16 1000.00 usec


F2 - Processing parametersSI 4096SF 500.1300038 MHzWDW SINESSB 2LB 0.00 HzGB 0PC 0.20

F1 - Processing parametersSI 1024MC2 echo-antiechoSF 500.1300038 MHzWDW SINESSB 2LB 0.00 HzGB 0

2D Gradient DQF-COSY2D Gradient DQF-COSY

33J=8.00HzJ=8.00Hz

TOCSYTOCSY ( (TOTOtal tal CCorrelationorrelation SSpectroscoppectroscopYY) or) orHOHAHAHOHAHA((HoHomonuclear monuclear HaHartman-rtman-HaHahn Spectroscopy)hn Spectroscopy)

t1 MLEV17 AQ

Pulse sequence for a TOCSY spectrum.

Different mixing time gives different degree of relay of Different mixing time gives different degree of relay of correlation. At small mixing time, TOCSY spectrum is similar to correlation. At small mixing time, TOCSY spectrum is similar to COSY spectrum. At long mixing time, gives total correlation.COSY spectrum. At long mixing time, gives total correlation.

HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

COSYCOSY RL-COSYRL-COSY TOCSYTOCSY

mixing timemixing time t2

ppm

1.01.52.02.53.03.5 ppm

1.0

1.5

2.0

2.5

3.0

3.5

Current Data ParametersNAME buttoc10EXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20001102Time 4.14INSTRUM DRX500PROBHD 5 mm TBI 1H/PULPROG mlevetgpTD 1024SOLVENT CDCl3NS 8DS 16SWH 2185.315 HzFIDRES 2.134096 HzAQ 0.2345700 secRG 16DW 228.800 usecDE 6.00 usecTE 300.0 KD0 0.00000300 secD1 1.79999995 secD9 0.01000000 secD11 0.03000000 secD16 0.00010000 secDELTA 0.00109943 secDELTA1 0.00110800 secFACTOR1 0IN0 0.00045765 secl1 0l3 128SCALEF 6

============ CHANNEL f1 =============NUC1 1HP1 5.60 usecP2 11.20 usecP5 19.33 usecP6 29.00 usecP7 58.00 usecP17 2500.00 usecPL1 -4.00 dBPL10 10.00 dBSFO1 500.1310815 MHz





HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

2D Gradient TOCSY, 2D Gradient TOCSY, mixing time=10ms

Same as COSY spectrumSame as COSY spectrum

11 22 33 4411

2233

44

1-21-22-32-3

3-43-4

ppm

1.01.52.02.53.03.5 ppm

1.0

1.5

2.0

2.5

3.0

3.5









11 22 33 4411

2233

44

1-21-22-32-3

3-43-4

1-31-3

2-42-4

Same as RLSame as RL －－ COSYCOSY

HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

ppm

1.01.52.02.53.03.5 ppm

1.0

1.5

2.0

2.5

3.0

3.5





F1 - Acquisition parametersND0 1TD 256SFO1 500.1311 MHzFIDRES 8.535453 HzSW 4.369 ppm


F1 - Processing parametersSI 1024MC2 echo-antiechoSF 500.1300144 MHzWDW QSINESSB 4LB 0.00 HzGB 0


11 22 33 4411

2233

44

1-21-22-32-3

3-43-4

1-31-3

2-42-41-41-4

HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

Total CorrelationTotal Correlation

DEPT: Distortionless Enhancement by Polarization Transfer

Heteronuclear expt.Detection: 13C

DistinguishCH, CH2, CH3

By suitable combination of =45, 90 & 135 spectra

All CH’s

Only CH

CH & CH3up CH2 down

HETCORHETCOR ( (HetHeteronuclear chemical shift eronuclear chemical shift corcorrelation, relation, 11H - H - 1313C COSY)C COSY)

13C

1H t1 1 2

The standard pulse sequence for The standard pulse sequence for 1313C-detectedC-detected11H-H-1313C chemical shift correlation.C chemical shift correlation.

AQt2

1H decouplingRemoving JCH splittings

*But Inverse experiment has the following Advantages:

•increase sensitivity of detecting the less sensitive nuclei

•1H is in the direct detection dimension => larger np => better resolution

HSQCHSQC ( (HHeteronuclear eteronuclear SSingle ingle QQuantum uantum CCoherence)oherence)

AQ

1H

13C /2 /2 t1 /2 /2 GARP-1

Pulse sequence for 1H-detected 1H-13C correlation through single-quantum coherence

• HSQC can give considerably better 13C resolution and sensitivity than HMQC for CH2 groups of natural products• HMQC has simpler pulse sequence and it is more robust and easier

to perform.

13C decouplingRemoving JCH splittings

HMQCHMQC ( (HHeteronuclear eteronuclear MMultiple ultiple QQuantum uantum CCoherence)oherence)

t2

ppm

1.01.52.02.53.03.5 ppm

10

15

20

25

30

35

40

45

50

55

60

65

Current Data ParametersNAME buthsqcEXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20001102Time 10.31INSTRUM DRX500PROBHD 5 mm TBI 1H/PULPROG invietgpsiTD 1024SOLVENT CDCl3NS 1DS 16SWH 2185.315 HzFIDRES 2.134096 HzAQ 0.2345700 secRG 2298.8DW 228.800 usecDE 6.00 usecTE 300.0 KD0 0.00000300 secD1 2.00000000 secD4 0.00170000 secD11 0.03000000 secD13 0.00000300 secD16 0.00010000 secD24 0.00090000 secDELTA 0.00116720 secDELTA1 0.00110700 secIN0 0.00003600 secl3 256

============ CHANNEL f1 =============NUC1 1HP1 5.60 usecP2 11.20 usecP28 2500.00 usecPL1 -4.00 dBSFO1 500.1310815 MHz

============ CHANNEL f2 =============CPDPRG2 garpNUC2 13CP3 17.70 usecP4 35.40 usecPCPD2 89.00 usecPL2 -1.00 dBPL12 13.00 dBSFO2 125.7633722 MHz





HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

11 22 33 44

1122

3344

HSQC spectrum: H-C correlatedHSQC spectrum: H-C correlated

(HMQC and HETCOR spectra look similar)(HMQC and HETCOR spectra look similar)

C-di

men

sion

H-dimensionH-dimension

HSQC-TOCSYHSQC-TOCSY

AQ

1H

13C /2 /2 t1 /2 /2 GARP-1

Pulse sequence for Pulse sequence for 11H-detected HSQC-TOCSY experimentH-detected HSQC-TOCSY experimentthrough single-quantum coherence.through single-quantum coherence.

Longer mixing time gives longer extend of spin-spin Longer mixing time gives longer extend of spin-spin correlations.correlations.

SLy

MLEV17

SLy

mixing timemixing time

ppm

1.01.52.02.53.03.5 ppm

10

15

20

25

30

35

40

45

50

55

60

65

Current Data ParametersNAME buthsto10EXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20001101Time 18.31INSTRUM DRX500PROBHD 5 mm TBI 1H/PULPROG invietgsmlTD 1024SOLVENT CDCl3NS 4DS 16SWH 2185.315 HzFIDRES 2.134096 HzAQ 0.2343412 secRG 2048DW 228.800 usecDE 6.00 usecTE 300.0 KCNST0 2.0000000D0 0.00000300 secD1 1.79999995 secD4 0.00170000 secD11 0.03000000 secD12 0.00002000 secD13 0.00000300 secD16 0.00010000 secd20 0.00111720 secd21 0.00110700 secIN0 0.00003600 secL1 3l3 128

============ CHANNEL f1 =============NUC1 1HP1 5.60 usecP2 11.20 usecP5 19.34 usecP6 29.00 usecP7 58.00 usecP17 2500.00 usecP28 2000.00 usecPL1 -4.00 dBPL10 10.00 dBSFO1 500.1310815 MHz


============ GRADIENT CHANNEL ========P16 1000.00 usec




HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

Same as Same as HSQCHSQC －－ COSYCOSY spectrum spectrum

Gradient HSQC-TOCSY(mixing time=10ms)Gradient HSQC-TOCSY(mixing time=10ms)

11 22 33 44

1122

3344

1-11-1

1-21-2

2-12-1

H-dimensionH-dimensionC-

dim

ensi

on

ppm

1.01.52.02.53.03.5 ppm

10

15

20

25

30

35

40

45

50

55

60

65









11 22 33 44

1122

3344

HO C

H1

H

C C C

H2

H

H3

H

H4

H

H


1-21-2

2-12-11-11-1

1-31-3

3-13-1

C-di

men

sion


ppm

1.01.52.02.53.03.5 ppm

10

15

20

25

30

35

40

45

50

55

60

65









HO C

H1

H

C C C

H2

H

H3

H

H4

H

H

11 22 33 44

1122

3344


1-21-2

2-12-11-11-1

1-31-3

3-13-1

1-41-4

4-14-1

C-di

men

sion


HMBCHMBC ( (HHeteronuclear eteronuclear MMultiple-ultiple-BBond ond CCorrelation Spectroscopy)orrelation Spectroscopy)

13C

1H t1 AQ

C2, C3 and C4: Quaternary or protonated carbons X: O, N

C1 C2 C3 C4

H1

C1 X C2 C3

H1

Pulse sequence for HMBCPulse sequence for HMBC

Long range connections or connections between spin systemsLong range connections or connections between spin systems

ppm

3.54.04.55.05.56.06.57.07.58.0 ppm

30

40

50

60

70

80

90

100

110

120

130

140

150

160

Current Data ParametersNAME cfyhmbcEXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20001103Time 7.36INSTRUM DRX500PROBHD 5 mm TBI 1H/PULPROG inv4gplrndTD 1024SOLVENT CDCl3NS 4DS 16SWH 3443.526 HzFIDRES 3.362818 HzAQ 0.1488800 secRG 11585.2DW 145.200 usecDE 6.00 usecTE 300.0 KD0 0.00000300 secD1 1.50000000 secD6 0.07000000 secD13 0.00000300 secD16 0.00050000 secIN0 0.00002380 sec


============ CHANNEL f2 =============NUC2 13CP3 15.70 usecPL2 -2.00 dBSFO2 125.7693867 MHz

============ GRADIENT CHANNEL ========GPNAM1 sine.100GPNAM2 sine.100GPNAM3 sine.100GPX1 0.00 %GPX2 0.00 %GPX3 0.00 %GPY1 0.00 %GPY2 0.00 %GPY3 0.00 %GPZ1 50.00 %GPZ2 30.00 %GPZ3 40.10 %P16 1000.00 usec



F1 - Processing parametersSI 512MC2 QFSF 125.7577961 MHzWDW QSINESSB 0LB 0.00 HzGB 0

N

N N

N

O

O

CH3

CH3

H3C

H

1

3

5

2

8

76

4

Gradient HMBCGradient HMBC

1122

3355

1144

3322

5566

7788

3-43-4

3-53-52-62-6

2-72-71-71-7

1-81-8

H-dimensionH-dimensionC-

dim

ensi

on

2D 2D NOESY NOESY ((NNuclear uclear OOverhauser verhauser EEnhancements nhancements SSpectroscoppectroscopYY))

t1 m AQ

The NOESY pulse sequence.

—C — ~ —C —

Ha HbVC*r -6, r0.5nm

r0.5nm

For resonance assignment, chemical structure elucidation & For resonance assignment, chemical structure elucidation & 3D structure determination3D structure determination

t2

ppm

3.54.04.55.05.56.06.57.07.5 ppm

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Current Data ParametersNAME cfynoesyEXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20001103Time 8.53INSTRUM DRX500PROBHD 5 mm TBI 1H/PULPROG noesygptpTD 1024SOLVENT CDCl3NS 8DS 16SWH 3443.526 HzFIDRES 3.362818 HzAQ 0.1488800 secRG 64DW 145.200 usecDE 6.00 usecTE 288.0 KD0 0.00000300 secD1 1.60000002 secD8 0.40000001 secD16 0.00010000 secd20 0.19890000 secIN0 0.00014520 sec


============ GRADIENT CHANNEL ========GPNAM1 sine.100GPNAM2 sine.100GPX1 0.00 %GPX2 0.00 %GPY1 0.00 %GPY2 0.00 %GPZ1 40.00 %GPZ2 -40.00 %P16 1000.00 usec



F1 - Processing parametersSI 1024MC2 TPPISF 500.1300144 MHzWDW SINESSB 2LB 0.00 HzGB 0

N

N N

N

O

O

CH3

CH3

H3C

H

1

3

5

2

8

76

4

3-CH3, 5-H3-CH3, 5-H

Gradient NOESYGradient NOESY

NOESY NOESY gives sequential gives sequential

assignment of peptidesassignment of peptides

23

12 3

CH2

CH2N C

H

N

O

H

C C

H

CH2O

N

H

C

CH2

C

H

OH

O

1 321

Spin-lock

90°

t1 t2

2D ROESY pulse program2D ROESY pulse program

For small molecule NOE can be very small or

zero ， ROESY can be used in place of NOESY

experiment. ROE intensity is also related to the H-H

distances.

mixing timemixing time

Structure Determination ProceduresStructure Determination Procedures

1D 1D 11H & H & 1313C & DEPT (+MSC & DEPT (+MS 、、 IRIR ，， basic chemical structure basic chemical structure

or or functional groups informationfunctional groups information ））Establish Establish 1313C-C-11H connections by thru bond JH connections by thru bond JCHCH couplin couplingsgs HMQCHMQC 、、 HSQCHSQC 、、 HSQC-TOCSY experiments HSQC-TOCSY experiments Establish Establish 11H-H-11H connection (spin systems or partial piH connection (spin systems or partial pieceseces ）） Decoupled Decoupled 11H, 1D TOCSY, 2D H, 1D TOCSY, 2D 11H-H-11H COSY, TOCSY expH COSY, TOCSY expts.ts. （（ usually starts with well-resolved usually starts with well-resolved 11H signalsH signals ））Long range connectionsLong range connections （（ connecting spin systems connecting spin systems & assigning & assigning quaternary carbonquaternary carbon ）） 1D NOESY & 2D HMBC, NOESY, ROESY experiments1D NOESY & 2D HMBC, NOESY, ROESY experiments3D structure or conformation determination3D structure or conformation determination 1D NOESY & 2D NOESY, ROESY, (HSQC)-NOESY expts.1D NOESY & 2D NOESY, ROESY, (HSQC)-NOESY expts.

NMR spectrum Structure Chem. Shift J Coupling Peak Int. NOE Correlation H-H ， C-H Applications: Sample quality control for Synthetic works. Elucidation.

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