Applications of NMR

Applications of NMR

MaterialChemistry

Biology

Medicine

Natural science

NMRNMR

Molecular Structure,Reaction Mechanism

Nucleic acid, RNA, DNA, and Protein

Analysis of soil, water, etc.

MRI (detecting the living cell), Metabolomics (metabolic fingerprint)

Molecular Structure,Property of matter

1D-NMR 1H spectrum

Deshielding (down field) Shielding (up field)

1. Chemical Shift

2. Spin-Spin Splitting(Coupling constant)

3. Integration

1. Local diamagnetic shieding (shilding/deshielding)• Electronegativity effect• Hybridization effect• Hydrogen bonding

2. Diamagnetic anisotropy • Ring current

1. Chemical Shift

Introduction to spectroscopy 4th, Pavia et al.

Chart for 1H Chemical Shift

Introduction to spectroscopy 4th, Pavia et al.

Chart for 13C Chemical Shift

Introduction to spectroscopy 4th, Pavia et al.

1. 2nI + 1 rule (n+1 rule for 1H and 13C)• Chemically equivalent / Magnetically equivalent

Homonuclear coupling Heteronuclear coupling

• Pascal’s triangle

2. Spin-Spin Splitting (Coupling constant)

Introduction to spectroscopy 4th, Pavia et al.

2. Coupling constant J

2. Spin-Spin Splitting (Coupling constant)

Introduction to spectroscopy 4th, Pavia et al.

Hz

Hz

ppm

Calculation of Coupling Constant

Calculation of Coupling Constant

Topspin (Bruker)Analysis Multiplet analysis Multiplet mode (mana)

1H / 13C Integration

• Molar concentrate ratio• The relative number of each type of atom• The mixture : detection of the relative amount of each compound

Standard• Inner standard • Insert tube with the standard• ERETIC

3. Integration

NMR tube

NMR tube

http://www.wilmad-labglass.com/

Experiment of Solvent Suppression

1H

Presaturation

NOESY

Watergate

Exitation sculpting

1H

NOESY

Presaturation Watergate

Exitation sculpting

Experiment of Solvent Suppression

13C NMR

NOE enhancement Without NOE enhancement

1H decoupled 13C-BB 13C-inverse gated

1H coupled 13C-1H coupled 13C

C CH CH2 CH3

DEPT 135 + - +DEPT 90 +DEPT 45 + + +13C-BB + + + +

13C NMR

Introduction to spectroscopy 4th, Pavia et al

Structure Analysis with NMR

Heteronuclear 2D: Through-bondHMQC, HSQC, HMBC

1D : 1H,13C, 19F, 29Si, 31P.. etc

Homonuclear 2D:Through-bondCOSY, TCOSY

Homonuclear 2D: Through-spaceNOESY, ROESY

COSY and TOCSY

COSY TOCSY

2 35 6

2 3

NOESY and ROESY Information

NOESY vs. ROESY cross peaks

NOESY ROESY

5 62 3

5 62 3

1H excitation, 1H observe: “INVERSE”• HSQC: heteronuclear single quantum coherence• HMQC: heteronuclear multiple quantum coherence• HMBC: heteronuclear multiple bond coherence

1H excitation, 13C observe: “OBSERVE”• XHCORR: X-H correlation• COLOC: X-H long-range correlation

Two types of heteronuclear experiments

5

6

2

3

1

7

4

HSQC HMBC

HSQC HMQC

Multiplicity-Edited HSQC

HMQC-TOCSY

• Selective COSY, TOCSY, NEOSY• Fast detection compared with 2D experiment• Quantitative results

Selective Experiment

Selective ExperimentSelective NOE

3-1

HONH2H

O HH

OH

L-tyrosine

1 2 4 56

75

6

3

3-1 3-2

Selective ExperimentSelective TOCSY

Complete Molecular Confidence (CMC)- Small molecule characterization and refinded small molecule analysis

Controlled Aggregation in Conjugated Polymer Nanoparticles via Organic Acid Treatments

O

O

O

O

4

4

* *

OO

NH2

O

OH2N

CPNs

Acetic acid Tartaric acid

Macromolecules 2011, 44, 5527–5530

Exp. 1

Absorbance and emission spectra of CPNs

• Acetic acid (blue dotted lines) and tartaric acid (red dotted lines) treatment CPNs in water and no treatment (black solid lines) CPNs in NMP

Exp. 1

1H NMR spectra of CPNs in D2O

Aromatic backbond range Side chain range

Exp. 1

1H NMR spectra of acetic acid treated CPNs in D2O

bc

de

f

gh

* *

O O

n

O O

a a

OO

O

NH2

O

H2N

O

O

3

3

i

CPNs

Exp. 1

NOESY spectra of acetic acid-treated CPNs in D2O

Exp. 1

Selective NOE spectra of acetic acid treated CPNs

7.36 ppm irradiation

7.05 ppm irradiation

Exp. 1

7.36 ppm / 7.05 ppm peak integration ratio plot of CPNs with various Concentration, Organic solvent (MeOD), and Temperature

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 0.5 1 1.5 2 2.5 3 3.5 4

Concentration (mg / mL)

7.36

 / 7.05 pp

m integ. ra

tio

Acetic acid ( ) and Tartaric acid ( ) treatment

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40 50

MeOD (%)

7.36

 / 7.05 pp

m integ. ra

tio

0

0.2

0.4

0.6

0.8

1

1.2

293 303 313 323 333

Temperature (K)

7.36

 / 7.05 pp

m integ. ra

tio

Concentration Organic solvent Temperature

Exp. 1

Interplay of aggregation CPNs.

Acetic acid (AA)treatment

Tartaric acid (TA)treatmentCPNs

Loose Aggregation Dense Aggregation

Exp. 1

1. Chemical shift 1H / 13C / 15N (HSQC / HMBC)

2. Kinetics

3. Relaxation delay T1 / T2 measurement

4. DiffusitonDOSY

5. EXSY

Dynamic experiments

Binding Site of ProteinBiochemistry 2013, 52, 1583−1593

Exp. 2

Kinetic Experiments

1h2h3h4h

5h6h7h

Lineweaver–Burk plot

Kcat (min-1), Enzymatic efficiency (mM-1min-1), sepcific activity (umol min-1 mg-1)

BAk

Diffusion coefficient D

DOSY

Self-assembly of octacyano-biscavitand by metal ligandinteraction: incorporation of container unit in polymer back bone

Tetrahedron Letters 47 (2006) 8847–8850

DOSY experiments

The concentration dependence of diffusion coefficients (x1010) of octacyano-biscavitand 2–Pd(dppp)OTf2 in CDCl3 at 298 K.

Exp. 3

Beilstein J. Org. Chem. 2012, 8, 90–99Exp. 4

Solution NMR

600MHz NMRCryoprobe z-gradient5mm TXI (1H /13C /15N)(15oC~50oC)

500MHz NMRz-gradient5 mm BBI ( 1H~109Ag) (15N, 17O, 29Si, 31P)(-100oC~100oC)

5 mm Dual High Temperature ( 1H/ 13C )(-100oC~240oC)

300MHz NMR5 mm QNP ( 1H/ 13C/ 19F/ 31P )(-100oC~100oC)

MAS = Magic Angle Spinning Line narrowing

CP = Cross polarization Signal enhancement : γI/ γS

γ 1H γ 13C x 4

Solid NMR

(3 cos 2 θ – 1) = 0 θ = 54.7°.

Abundant spins (1H)

Rare spins (13C)

CPMAS / CPTOSS

CPMAS5KHz spinning

CPMAS11KHz spinning

CPTOSS5KHz spinning

NQS & CPPI

11KHz spinning

5KHz spinning

CPMAS-CPPI

CPMAS-NQS

CPMAS

CPTOSS-NQS

CPTOSS

13C NMR of tyrosine

500MHz Solid NMR4mm probe

2hr

600MHz NMR10hr

500MHz solid NMR

• 2.5mm probe head - Spinning rate : ~ 35 kHz (-30oC ~ 70oC) / 14uL

• 4mm probe head- Spinning rate : ~ 15 kHz (-140oC ~ 150oC) /50uL

• 7mm probe head - Spinning rate : ~ 7 kHz (-140oC ~ 150oC) /230uL

( 11B, 13C, 15N, 17O, 19F, 23Na, 27Al , 29Si, 31P )

• Pulse field gradient probe for diffusion• Low Temperature probe and cryostat( 4K ~ 350K )