Structure Elucidation through NMR Spectroscopy. Dr. Amit Kumar Yadav Assistant Professor-Chemistry Maharana Pratap Govt. P.G.

Transcription

1 Structure Elucidation through NMR Spectroscopy Dr. Amit Kumar Yadav Assistant Professor-Chemistry Maharana Pratap Govt. P.G. College, ardoi

2 Introduction NMR is the most powerful tool available for organic structure determination. It is used to study a wide variety of nuclei: 1 13C 15N 19F 31P

3 Nuclear Spin A nucleus with an odd atomic number or an odd mass number has a nuclear spin. The spinning charged nucleus generates a magnetic field.

4 External Magnetic Field When placed in an external field, spinning protons act like bar magnets.

5 Two Energy States The magnetic fields of the spinning nuclei will align either with the external field, or against the field. A photon with the right amount of energy can be absorbed and cause the spinning proton to flip.

6 E and Magnet Strength Energy difference is proportional to the magnetic field strength. E = h = h B0 2 Gyromagnetic ratio,, is a constant for each nucleus (26,753 s-1gauss-1 for ). In a 14,092 gauss field, a 60 Mz photon is required to flip a proton. Low energy, radio frequency.

7 Magnetic Shielding If all protons absorbed the same amount of energy in a given magnetic field, not much information could be obtained. But protons are surrounded by electrons that shield them from the external field. Circulating electrons create an induced magnetic field that opposes the external magnetic field.

8 Shielded Protons Magnetic field strength must be increased for a shielded proton to flip at the same frequency.

9 The NMR Spectrometer

10 Tetramethylsilane 3 C C 3 Si C 3 C 3 TMS is added to the sample. Since silicon is less electronegative than carbon, TMS protons are highly shielded. Signal defined as zero. rganic protons absorb downfield (to the left) of the TMS signal.

11 Chemical Shift Measured in parts per million. Ratio of shift downfield from TMS (z) to total spectrometer frequency (z). Same value for 60, 100, 300 Mz or above machines. Symbolized as delta scale.

12 Electronegativity Factors Influencing Chemical Shift o Electronegativity can be a guide to chemical shift only up to a point where other effects are not operating. Degree of shielding depends on the density of the circulating electrons on the particular nucleus which will directly depend on the inductive effect of the attached groups. o More electronegative atoms deshield more and give larger shift values. o Effect decreases with distance. o Additional electronegative atoms cause increase in chemical shift.

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14 Van Der Waals Deshielding o Steric hinderence causes electrostatic repulsion which will tend to repel the electron surrounding the proton. o The proton will be deshielded and appear at higher value. (less than 1 ppm), o Must be taken in to account while predicting chemical shifts of overcrowded molecules like steroids, triterpenoids, alkaloids etc. Anisotropic effects o This effect depends on the diamagnetic anisotropy, which means that shielding and deshielding depend on the orientation of the molecule with respect to the applied magnetic field i.e the effects are paramagnetic in certain directions around the clouds and diamagnetic in others hence anisotropic as opposed to isotropic (operating equally through space).

15 Aromatic Protons, 7-8 Vinylic Protons, 5-6 Acetylenic Protons, 2.5 Aldehydic Protons, 9-10

16 Spin-Spin Splitting Nonequivalent protons on adjacent carbons have magnetic fields that may align with or oppose the external field. This magnetic coupling causes the proton to absorb slightly downfield when the external field is reinforced and slightly upfield when the external field is opposed. All possibilities exist, so signal is split.

17 The N + 1 Rule If a signal is split by N equivalent protons, it will split into N + 1 peaks.

18 Range of Magnetic Coupling Equivalent protons do not split each other. Protons bonded to the same carbon will split each other only if they are not equivalent. Protons on adjacent carbons normally will couple. Protons separated by four or more bonds will not couple. Coupling Constants Distance between the peaks of multiplet measured in z Not dependent on strength of the external field Multiplets with the same coupling constants may come from adjacent groups of protons that split each other.

19 Values of coupling constant

20 Complex Splitting Signals may be split by adjacent protons, different from each other, with different coupling constants. Example: a of styrene which is split by an adjacent trans to it (J = 17 z) and an adjacent cis to it (J = 11 z). a C C c b

21 The Karplus equation The most common coupling constant we ll see is the three bond coupling, or 3J: As with the 1J or 2J, the coupling arises from the interactions between nuclei and electron spins. 1J and 3J will hold the same sign, while 2J will have opposite sign. owever, the overlap of electron and nuclear wavefunctions in the case of 3J couplings will depend on the dihedral angle <φ> formed between the C vectors in the system. The magnitude of the 3J couplings will have a periodic variation with the torsion anlge, something that was first observed by Martin Karplus in the 1950 s.

22 The magnitude of the 3 J couplings will have a periodic variation with the torsion anlge, something that was first observed by Martin Karplus in the 1950 s. The relationship can be expressed as a cosine series: A, B, and C are constants that depend on the topology of the bond (i.e., on the electronegativity of the substituents). Graphically, the Karplus equation looks like this: A nice feature of the Karplus equation is that we can estimate dihedral angles from 3 J coupling constants. Thus, a variety of A, B, and C parameters have been determined for peptides, sugars, etc., etc.

23 Carbon-13 12C has no magnetic spin. 13C has a magnetic spin, but is only 1% of the carbon in a sample. The gyromagnetic ratio of 13C is one-fourth of that of 1. Signals are weak, getting lost in noise. undreds of spectra are taken, averaged.

24 1 and 13 C Chemical Shifts =>

25 2D NMR Techniques 1. omonuclear CSY (Correlation spectroscopy): 1-1 CSY (scalar coupling i.e. δ δ correlation spectroscopy) J-Resolved spectroscopy (M 2DJ): one axis contains δ values which are correlated to J values on the other axis. CSY-45: Modification of CSY to reduce the intensity of the diagonal signals with respect to the intensity of cross peaks. Possible overlaps can thus be avoided. DQF-CSY (Double quantum filter): Provides better visualization of cross peaks nearer to diagonal. This sequence preferentially attenuates the single quantum resonances of the diagonal with respect to cross peaks and also suppresses the detection of isolated protons such as those arising from solvent or isolated methyl groups i.e. magnetization of singlet signals is suppressed. TQF-CSY (triple quantum filter) : All the spin systems that contain less than three or more mutually coupled spins are eliminated by use of TQF. Example of such system is; exopyranosides where -5, -6 and -6' cross peaks were eliminated in TQF-CSY spectrum. Rings containing equatorial protons prevent coherence or polarization transfer fron -1 to -5 and -6 in RELAY experiments.

26 PS-CSY (Phase sensitive): The basis for achieving remote connectivities. Crtoss peaks obtained at F-2 (horizontal axis) of one and F-12 (vertical axis) of another proton not only showed coupling between themselves (active coupling) but also with other protons (passive coupling). Cross section through one peak parallel to f-2 shows multiplet pattern of this resonance and vice-versa for F-1 plane. J2,3, J3,4, J4,5ax are in the range 8-10 z for diaxial gluco. where as for galacto confor. J4,5 is 2 z. Similarily they can be useful for identification of anomeric conformation in gluco, galacto or manno configurations.

27 1 NMR spectrum

28 13C NMR spectrum

29 DQF-CSY spectrum

30 2. eteronuclear Correlation spectroscopy ETCR (13C-1 eteronuclear Correlation Spectroscopy) Each cross peak arises from connectivity between a 13C nucleus and its directly bonded protons having the coordinates (δc, δ). (It must be mentioned that in such experiments only 1 % of the protons which are coupled to 13C are actually detected (due to less abundance of 13C nuclei) as compared to 1 detected experiments.) SQC (eteronuclear single quantum coherence) MQC (eteronuclear multiple quantum coherence) SQC and MQC both are 1 detected experiments and provide one bond correlation with high resolution in 13C domain. CLC (Correlation via Long range Coupling): 13C detected experiment where long range 13C, 1 couplings are observed. MBC (eteronuclear multiple-bond correlations): 1 detected experiments superior to CLC by its reliability in long range correlations. Long range three bond correlations appear in the spectrum which facilitates in determining the remote connectivities in structural framework.

31 Gradient SQC spectrum of 2a

32 Gradient MBC spectrum

33 3. RELAY Experiments These experiments are based on polarization transfer phenomenon. In this approach whole coupling network can be translated. AA (omonuclear artmann-ann Spectroscopy) or TCSY (Total Correlation Spectroscopy): The most useful method of relay of coherence along the chain of spins is the isotopic mixing experiments in which the net magnetization is transferred under spin locking. elpful in determining the J-network (group of protons serially linked via 1 1 J (scalar) couplings. Requirement of the experiment is presence of at least one well resolved signal of the J-network (as anomeric proton in the case of oligosaccharides) During spinlock or mixing time magnetization transfer takes place and short mixing time (20-50 ms) leads primarily cross peaks of strongly coupled protons while longer mixing time ( ms) allows magnetization transfer to remote protons of the spin system.

34 1D TCSY spectrum R 1 R 1 R 1 1'' 2'' R 1 R 1 R 1 2' 1' R 1 R R 1 R 2 R 2 R 1 = Bn, R 2 R 2 = Benzylidene

35 TCSY spectrum xopro Sar N-MeVal Val Thr Pro Sar N-MeVal Val Thr N N 2 ppm 1 C 3 C ppm

36 4. Dipolar Couplings: NESY (Nuclear verhauser effect spectroscopy): NE connectivities are often observed between signals which are close oriented in the three dimensional space. The presence of inter-residue NE from the anomeric ptoton of a particular sugar residue to protons of the other sugar residues in case of oligosaccharides or to non sugar residues in case of glycosides, defines the glycosidic linkage between the two residues. It depends upon spatial proximity of the protons. RESY (NE in Rotating frame): Due to problems with NE measurements at medium field strength in NESY, RESY can produce reliable NE cross peaks not obtained from NESY.

37 RESY spectrum C 3-20 C C-12-6 C a C 3-17

38 RESY spectrum of the epimer 6-CC 3 C 3-20 C C-12 C C a

39 R MAS NMR igh-resolution magic-angle spinning (RMAS) probes allow NMR spectra to be collected on a wide range of heterogeneous samples primarily because they average the inhomogeneity created by magnetic susceptibility differences. Tissue homogenates, soil samples, whole cells, solid phase organic synthesis and the study of chromatographic stationary phases are a few applications where RMAS NMR probes have found widespread usage. ne reported application that has received very little attention is the use of RMAS NMR to characterize compounds directly from separated thin-layer chromatography (TLC) spots. In a preliminary report, Wilson et al. demonstrated that NMR spectra could be obtained for model compounds separated by reverse-phase TLC (RPTLC) simply by removing the separated spots from the RPTLC plate, transferring the dry powder containing the adsorbed analyte to an RMAS sample rotor, and forming a slurry with D2. Wilson ID, Spraul M, umpfer E. J. Planar Chromatogr. Mod. TLC 1997; 10: 217.

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