Nmr Spectroscopy Chemistry 330

2 Objectives u Nmr basics u chemical shifts u relaxation times u 2-Dimensional Nmr experiments u COSY u NOESY u What kind of information do we obtain?

3 Nmr Basics u The signal in a nmr spectrum arises from transitions between nuclear spin states. u 1 H, 13 C, 31 P all have a nuclear spin quantum number, I = 1/2. u The total number of spin states 2I + 1 = 2

4 Energy Levels in the Spin 1/2 System  -1/2  +1/2

5 The Nmr Signal  (Hz)

6 The Magnetisation Vector u We can represent the slight excess of spins in the  state by use of the nuclear magnetization vector, M o x y z

7 Shielding u The presence of the bonding electrons about the nucleus gives rise to ‘electron shields’ around the nucleus u Shielding constant 

8 The Nmr Spectrum with Shielding  (Hz) 

9 The Chemical Shift u Resonance frequencies are field dependent u Define a field-independent parameter - the chemical shift (  ) u o - magnet strength in MHz

10 The Chemical Shift Spectrum  (ppm) 

11 J-Coupling (spin-spin splitting) u What happens when we have non- equivalent protons on adjacent C atoms?

12 The Coupling Constant  (ppm)  J ab (Hz)

13 The COSY Experiment u COrrelation SpectroscopY u What happens when we project the following spin system in two-dimensions? J ab  J bc HaHa HbHb HcHc

14 The COSY Spectrum  (ppm)

15 The NOESY Experiment u In the 2-D NOESY (nuclear Overhauser enhancement) experiment, we look for ‘through space’ dipolar couplings. HaHa HbHb HcHc J ab > 0 J ac = 0

16 The NOESY Spectrum  (ppm)

17 The Origin of Spin-Lattice Relaxation u By using a selective pulse (a 180  pulse), we can invert the populations of the nuclear spin states y x z

18 Spin-Lattice Relaxation u The spin system attempts to re-establish the equilibrium magnetisation vector. We observe the magnitude of the magnetisation vector as a function of time after the inversion pulse is applied. (180  -  n - 90  - acquire) n

19 11 y x z

20 22 y x z

21 33 y x z

22 44 y x z

23 55 y x z

24 66 y x z

25 77 y x z

26 88 y x z

27 Magnetisation Intensity vs. Time MM

28 The Spin Lattice Relaxation Time u The time constant governing the decay process is the spin-lattice relaxation time, T 1 u M  = limiting value of magnetisation intensity u M  = magnetisation intensity at t = 

29 Nuclear Overhauser Enhancement (NOESY) Experiments u 2-D Nmr NOESY experiments have been used extensively in the determination of the conformations of nucleic acids, proteins, and membranes. u The presence of the cross peak in the 2-D NOESY spectrum indicates the presence of intermolecular or intramolecular dipolar interactions (representing a spatial proximity of < 0.50 nm).

30 NOESY Spectrum for SDS/C 4 OH

31 NOESY Spectrum for SDS/Bz

32 NOESY Spectrum for DTAB/Bz