NMR Spectroscopy in Notre Dame

Transcription

1 NMR Spectroscopy in Notre Dame University of Notre Dame College of Science Department of Chemistry and Biochemistry Nuclear Magnetic Resonance Facility

2 Reservation system for spectrometers

3 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.

4 NMR and MRI Instruments Varian-301: 2 rf channels, probe 5 mm 1 H/ 19 F/ 13 C/ 31 P, VT -100 to +150 o C Varian-302: 2 rf channels, probe 5 mm broadband, VT -100 to +150 o C Bruker-400: 2 rf channels, probe 5 mm broadband z-pfg 11 B free, VT -80 to +130 o C 4 mm HR MAS z-pfg, VT -20 to +70 o C Bruker-401: 2 rf channels, probe 5 mm broadband z-pfg, VT -80 to +130 o C Varian-500: 4 rf channels, probe 5 mm broadband, VT -100 to +150 o C 5 mm 1 H/ 13 C/ 15 N xyz-pfg, VT 0 to +50 o C Varian-600: 3 rf channels, probe 5 mm AutoX broadband z-pfg, VT -80 to +130 o C 5 mm 1 H/ 13 C/ 15 N- 31 P z-pfg, VT -100 to +130 o C Bruker-700: 4 rf channels, cryoprobe 5 mm 1 H/ 13 C/ 15 N z-pfg, VT 0 to +60 o C Bruker-800: 4 rf channels, cryoprobe 5 mm 1 H/ 13 C/ 15 N z-pfg, VT 0 to +60 o C probe 5 mm broadband z-pfg, VT -150 to +150 o C Bruker MRI 300: 2 rf channels, micro-pfg coil 100 G/cm with 2.5, 5, 10, 15, 20, 25, 30, 35 mm 1 H rf coils mini-pfg coil 14 G/cm with 60 mm 1 H rf coil Dr. I. Veretennikov, 143 NSH, , ivereten@nd.edu

5 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.

6 Parameters used for structure determination: chemical shifts, δ scalar spin-spin coupling constants, J vicinal coupling constants 3 J = A. cos 2 φ + B. cosφ + C, Karplus equation, φ - dihedral angle integrals nuclear Overhauser effect, noe residual dipolar coupling constants, D D ~ (3. cos 2 θ 1), θ angle between internuclear vector and B 0 Parameters used for investigation of molecular dynamics: spin-lattice (longitudinal) relaxation time, T 1 dipolar, quadrupolar, chemical shift anisotropy, scalar, spin-rotation spin-spin (transverse) relaxation time, T 2, chemical exchange spin-lattice relaxation time in the rotating frame, T 1ρ, chemical exchange nuclear Overhauser effect, noe diffusion constants, D dif DOSY NMR, mixture, molecular weight, geometry, complexation

7 Some NMR active atomic nuclei used for studies of organic molecules: 1 H, 13 C, 31 P, 2 H, 19 F, and 15 N. Practical aspects of acquiring NMR spectra Sample preparation NMR instrumentation Data acquisition and processing - 1D experiments - 2D experiments Verification of proposed structure Example nitrocefin, analysis of 1D and 2D spectra Unknown compound

8 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 ( ml for 5 mm tube, sample height ~5-6 cm) Example: 20 mm sample with 1.4 mm impurity Sample S s /N = 100/1 in 30 sec S/N ~ n. c S i /N = 100. c i /c s c s sample concentration c i impurity concentration Impurity S i /N = 7/1 Time needed for impurity S i /N = 100/1 7 x c s /c i, ~49 min

9 Components of current NMR spectrometers - Superconducting magnets T MHz - Electronic consoles with components utilizing digital technology, 2-4 rf channels - Computers PCs with Linux or Windows - Probes broadband, inverse, multinuclear, cryoprobes; with or without PFG; with inner diameter 1.7, 3, 5, 10 mm; microprobes µl; flow-through

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11 NMR Probe Coils VT gas Tuning Matching Capacitors

12 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: FID B 0 Heteronuclear:

13 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 Hz np=4096, at=0.893 s 16384, s 65536, s Relaxation delay, d1 Ernst angle α=arccos(exp(-(at+d1)/t 1 ))

14 Processing parameters for 1D spectra Number of points used in FT, fn fn > np zero filling Apodization, multiplication of the FID signal by a mathematical function exponential: g(t) = exp(-a. t) gaussian: g(t) = exp(a. t). exp(-b. t 2 ) trigonometric functions sin and cos; sin: g(t) = sin((π-c). ((t/aq)+c) A) zero filling B) exponential C) sin 90 O shifted D) sin 70 O shifted E) sin 50 O shifted

15 Acquisition parameters for 2D spectra Generalized pulse sequence for 2D experiments acquisition time at 1 =p/sw1 acquisition time at 2 =r/sw2 time domain t 2 frequency domain F 2 spectral window sw2 (sw) time domain t 1 frequency domain F 1 spectral window sw1 Homonuclear spectra sw1=sw2; diagonal and non diagonal (cross) peaks Heteronuclear spectra sw1 sw2; only crosspeaks

16 Processing 2D spectra Linear prediction forward or backward no more than double of number of acquired points x n = a 1. x n-1 + a 2. x n a m. x n-m Apodization Zero filling Magnitude spectra 4 coefficients for FT (Fourier Transformation) Phase sensitive spectra 8 coefficients for FT

17 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 SpectroscopY TOCSY - TOtal Correlation SpectroscopY Proton - Carbon Connectivities: HSQC - Heteronuclear Single Quantum Correlation HMQC - Heteronuclear Multiple Quantum Correlation HETCOR - HETeronuclear CORrelation HMBC - Heteronuclear Multiple Bond Correlation HETLOC - HETeronuclear LOng-Range Coupling HETCOR: F 2 and sw2 correspond to carbon frequencies (chemical shifts) F 1 and sw1 correspond to proton frequencies HMQC, HSQC, HMBC: F 2 and sw2 correspond to proton frequencies F 1 and sw1 correspond to carbon frequencies

18 Experiments Utilizing Through Space Dipolar Interactions Proton - Proton Connectivities: NOESY - Nuclear Overhauser Effect SpectroscopY ROESY - Rotational Overhauser Effect SpectroscopY NOE ~ 1/r 6 5 Å Intramolecular NOE Presence of paramagnetic impurities weakens intensity of NOE EXSY 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.

19 Magnitude Spectrum: COSY

20 diagonal peak crosspeak

21 Phase sensitive spectrum: HSQC

22 positive crosspeak negative crosspeak

23 Verification of nitrocefin molecular structure

24 strongly coupled dd, 1.3, 15.6 Hz dddd, ~1.3, 3.1 Hz dd, 3.1, 5.1 Hz d, 16.1 Hz d, 16.1 Hz

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26 COSY H-6 H-7 NH H-4 H-2 H-13 H-14 TOCSY

27 HSQC CH-7 CH-6 CH 2-2 CH 2-10 CH-7 CH-5 CH-4 CH-1 CH-2 CH-14 CH-12 CH-13 C-9 HMBC C-8

28 HMBC C-11 CH-12 CH-1 C-4 C-3 HMBC C g -8 C-6 C-3

29 ROESY ROESY H-4,H1

30 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 (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 1 H and 13 C{ 1 H} spectra, 2D COSY, TOCSY, HSQC (HMQC, HETCOR), HMBC, ROESY spectra