NMR Spectroscopy in Notre Dame

University of Notre Dame

College of Science

Department of Chemistry and Biochemistry

Nuclear Magnetic Resonance Facilityhttp://www.nd.edu/~nmr

Reservation system for spectrometers

Safety Rules

Before beginning work in the NMR facility laboratories, the following safety rules must be observed.

• Person with medical devices such as cardiac pacemakers and prosthetic parts must remain outside the 5-gauss perimeter.

• Metal object must remain outside the 5-gauss perimeter. Metallic paper clips and staples should not be brought in the labs.

• In the event of a “magnet quench” leave the area immediately, leaving the doors to the NMR lab open.

• Do not look down the magnet upper barrel if probe is in the place.• Cards with magnetic strips (ATM, credit, driver’s licenses) should

remain outside the 5-gauss perimeter. • Do not exceed boiling or freezing points of a sample. • Be very careful with sample tubes.

NMR and MRI Instruments

• Varian-301: 2 rf channels, probe 5 mm 1H/19F/13C/31P, VT -100 to +150 oC

• Varian-302: 2 rf channels, probe 5 mm broadband, VT -100 to +150 oC

• Bruker-400: 2 rf channels, probe 5 mm broadband z-PFG 11B free, VT -80 to +130 oC4 mm HR MAS z-PFG, VT -20 to +70 oC

• Bruker-401: 2 rf channels, probe 5 mm broadband z-PFG, VT -80 to +130 oC

• Varian-500: 4 rf channels, probe 5 mm broadband, VT -100 to +150 oC5 mm 1H/13C/15N xyz-PFG, VT 0 to +50 oC

• Varian-600: 3 rf channels, probe 5 mm AutoX broadband z-PFG, VT -80 to +130 oC5 mm 1H/13C/15N-31P z-PFG, VT -100 to +130 oC

• Bruker-700: 4 rf channels, cryoprobe 5 mm 1H/13C/15N z-PFG, VT 0 to +60 oC

• Bruker-800: 4 rf channels, cryoprobe 5 mm 1H/13C/15N z-PFG, VT 0 to +60 oCprobe 5 mm broadband z-PFG, VT -150 to +150 oC

• Bruker MRI 300: 2 rf channels, micro-PFG coil 100 G/cm with 2.5, 5, 10, 15, 20, 25, 30, 35 mm 1H rf coilsmini-PFG coil 14 G/cm with 60 mm 1H rf coilDr. I. Veretennikov, 143 NSH, 1-7601, ivereten@nd.edu

Determination of Structure and Dynamics of Organic Molecules in Solutions by NMR Spectroscopy

Techniques

The uniqueness of NMR spectroscopy techniques, when applied to studies of organic molecules, lies in the

fact that they can be used for both establishing molecular structure and investigation of molecular

dynamics.

http://www.nd.edu/~nmr

• chemical shifts, δ• scalar spin-spin coupling constants, J

vicinal coupling constants 3J = A.cos2φ + B.cosφ + C, Karplus equation, φ - dihedral angle• integrals• nuclear Overhauser effect, noe• residual dipolar coupling constants, D

D ~ (3.cos2θ – 1), θ – angle between internuclear vector and B0

Parameters used for structure determination:

Parameters used for investigation of molecular dynamics:

• spin-lattice (longitudinal) relaxation time, T1dipolar, quadrupolar, chemical shift anisotropy, scalar, spin-rotation

• spin-spin (transverse) relaxation time, T2, chemical exchange

• spin-lattice relaxation time in the rotating frame, T1ρ, chemical exchange

• nuclear Overhauser effect, noe• diffusion constants, Ddif

DOSY NMR, mixture, molecular weight, geometry, complexation

Practical aspects of acquiring NMR spectra• Sample preparation• NMR instrumentation• Data acquisition and processing

- 1D experiments- 2D experiments

Some NMR active atomic nuclei used for studies of organic molecules:

1H, 13C, 31P, 2H, 19F, and 15N.

Verification of proposed structure

• Example nitrocefin, analysis of 1D and 2D spectra• Unknown compound

Sample preparation

• NMR tube selection (Wilmad PP-528, PP-541; Shigemi)• Solvent selection (dilute samples – higher degree solvent deuteration) • Sample purity and concentration (optimal ~20-50 mM, avoid >100 mM)• Sample volume (0.6-0.7 mL for 5 mm tube, sample height ~5-6 cm)

Example: 20 mM sample with 1.4 mM impuritySample Ss/N = 100/1 in 30 secS/N ~ √n . c → Si/N = 100 . ci/cscs – sample concentration ci – impurity concentrationImpurity Si/N = 7/1

Time needed for impurity Si/N = 100/1 7 x cs/ci, ~49 min

Components of current NMR spectrometers

- Superconducting magnets4.70 – 21.14 T 200 – 900 MHz

- Electronic consoles with components utilizing digitaltechnology, 2 - 4 rf channels

- ComputersPCs with Linux or Windows

- Probes broadband, inverse, multinuclear,cryoprobes; with or without PFG; with inner diameter 1.7, 3, 5, 10mm; microprobes 40–80 µL;flow-through

NMR Probe

Coils

CapacitorsTuningMatching

VT gas

Data Acquisition and Processing

Values of acquisition and processing parameters for individual experiments are loaded by the corresponding set up commands.

Pulse sequences for measurements of 1 D spectra

Homonuclear:

Heteronuclear:

FID B0

Important acquisition parameters for 1D spectra

• Spectral width (window), sw• Frequency corresponding to the center of spectral window, tof • Number of points used for digitization of FID (Free Induction Decay)

signal, np acquisition time at=np/sw, dwell time dw=1/sw

Example: sw = 4588 Hznp=4096, at=0.893 s

16384, 3.571 s 65536, 14.284 s

• Relaxation delay, d1Ernst angle α=arccos(exp(-(at+d1)/T1))

Processing parameters for 1D spectra

• Number of points used in FT, fnfn > np zero filling

• Apodization, multiplication of the FID signal by a mathematical functionexponential: g(t) = exp(-a.t) gaussian: g(t) = exp(a.t) . exp(-b.t2)trigonometric functions sin and cos; sin: g(t) = sin((π-c).((t/aq)+c)

A) zero filling

B) exponential

C) sin 90O shifted

D) sin 70O shifted

E) sin 50O shifted

Acquisition parameters for 2D spectra

Generalized pulse sequence for 2D experiments

time domain t2 frequency domain F2 spectral window sw2 (sw)time domain t1 frequency domain F1 spectral window sw1Homonuclear spectra sw1=sw2; diagonal and non diagonal (cross) peaks Heteronuclear spectra sw1≠sw2; only crosspeaks

acquisition time at1=p/sw1acquisition time at2=r/sw2

Processing 2D spectra

• Linear prediction forward or backward no more than double of number of acquired points xn = a1

.xn-1 + a2.xn-2 + …. + am

.xn-m

• Apodization• Zero filling• Magnitude spectra 4 coefficients for FT (Fourier Transformation)• Phase sensitive spectra 8 coefficients for FT

2D NMR Experiments for Establishing Proton-Proton and Proton-Carbon Connectivities

Experiments Utilizing Spin-Spin Interactions

Proton - Proton Connectivities:COSY - COrrelation SpectroscopY DQFCOSY - Double Quantum Filtered COrrelation SpectroscopYTOCSY - TOtal Correlation SpectroscopY

Proton - Carbon Connectivities:HSQC - Heteronuclear Single Quantum CorrelationHMQC - Heteronuclear Multiple Quantum CorrelationHETCOR - HETeronuclear CORrelationHMBC - Heteronuclear Multiple Bond CorrelationHETLOC - HETeronuclear LOng-Range Coupling

HETCOR: F2 and sw2 correspond to carbon frequencies (chemical shifts)F1 and sw1 correspond to proton frequencies

HMQC, HSQC, HMBC: F2 and sw2 correspond to proton frequenciesF1 and sw1 correspond to carbon frequencies

Experiments Utilizing Through Space Dipolar Interactions

Proton - Proton Connectivities:NOESY - Nuclear Overhauser Effect SpectroscopYROESY - Rotational Overhauser Effect SpectroscopY

NOE ~ 1/r6 ≲5 Å Intramolecular NOEPresence of paramagnetic impurities weakens intensity of NOEEXSY experiment

1D NOE experiment - NOE difference

To successfully analyze NOE experiments in terms of molecular structure, unambiguous assignment of the proton resonance signals must be obtained first by measuring spectra utilizing scalar couplings.

Magnitude Spectrum: COSY

diagonal peak → ← crosspeak

Phase sensitive spectrum: HSQC

negative crosspeak →

positive crosspeak →

Verification of nitrocefin molecular structure

dddd, ~1.3, 3.1 Hzdd, 1.3, 15.6 Hz

d, 16.1 Hz d, 16.1 Hz

strongly coupled

dd, 3.1, 5.1 Hz

COSY

TOCSY

H-7 →

H-6 →

NH ↓

↖ H-14’

H-13’↓

↖ H-4’

H-2’ ↘

HSQC

HMBC

↑CH2-10’

↑CH-7

CH-6↓

CH-7’↓ CH-5’

↓CH-4’

↓CH-1’

↓

CH-2’↓

↑CH-14’

CH-12’↓

↖ CH-13’

CH2-2 ↗

↖ C-9’

← C-8

HMBC

HMBC

C-11’ →

CH-12’↓

↑CH-1’

↑C-4

↑C-3

Cg-8’ ↘ ↙ C-6’ C-3’ ↘

← H-4’,H1’

ROESY

ROESY

Elucidation Unknown Molecular Structures

As much information as possible should be gathered about the unknown structure before NMR studies.

• Molecular formula (elemental analysis or high resolution MS spectrometry)

• Identification of functional groups (IR, UV/Vis)

• Calculation of the degree of unsaturation, U, (number of rings and multiple bonds)U = C + 1 – 0.5.(H+X-N)

C and H are numbers of carbon and hydrogen atoms, respectively. X and N are numbers of heteroatoms with valence 1 and 3, respectively.

• Measurement and analysis of 1D 1H and 13C{1H} spectra, 2D COSY, TOCSY, HSQC (HMQC, HETCOR), HMBC, ROESY spectra