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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 )