Elucidation of Organic Structures II.

NMR spectroscopy

The physical basis of the Nuclear Magnetic Resonance experiment:

- The origin of the NMR signal by pulse excitation. Acquisition and Fourier transformation. The Bloch equation. The spin-spin decoupling and saturation.

- Relaxation effects. The spin-lattice (T₁) and spin-spin relaxation time (T₂)

- Measurement techniques to determine the relaxation times. (Inversion recovery, spin-echo).

- The connection of the relaxation time data and molecular mobility.

- Application of magnetic field gradients in NMR. The fundamental aspects of the MRI (Magnetic resonance imaging).

- Heteronuclear NMR spectroscopy: Applications of the ³¹P-NMR spectroscopy in chemistry and biochemistry.

4 hours

- One-dimensional pulse techniques and their applications.

- The homonuclear and heteronuclear polarization transfer. Application at the measurements of rare-spin nuclei: SPI, INEPT, refocussed INEPT, DEPT. Application of adiabatic pulses: Q-DEPT. Spectral editing.

- The Nuclear Overhauser effect. (NOE). The Solomon equation and its interpretation.

- NOE measurement techniques. 1D NOE-difference spectra and application in determination of the molecular structure. Examples.

- Heteronuclear NMR spectroscopy: Applications of ²H- and ²³Na-NMR spectroscopy in chemistry and biochemistry.

4 hours

Two-dimensional NMR spectroscopy and applications:

- Principles of two-dimensional NMR spectroscopy. The basic steps: preparation, evolution, mixing, acquisition.

- Shift-correlated two-dimensional methods, H/H and C/H-correlations.

- Measurement of the NOE in two dimension: the NOESY experiment. Interpretation of NOESY data, correlation with molecular modelling results.

- Exchange spectroscopy. Application of the EXSY spectra in the study of rate processes.

- Experiments using spin-lock pulses (TOCSY, ROESY).

- Proton-detected heteronuclear NMR methods. HMQC, HSQC, HMBC, H2BC.

- Homonuclear and heteronuclear J-spectroscopy.

- Diffusion spectroscopy (DOSY).

4 hours

Solid state NMR: chemical shifts anisotropy, line broadening effects of homo- and heteronuclear couplings. Effects of magic angle spinning (MAS) and spinning rate. ¹H → ¹³C cross polarisation. HR-CP-MAS technique. ¹³C applications: study on polymorph samples. 2D HR-CP-MAS measurements. Enhancement in the F₁ (¹H) dimension.

4 hours

Enhancement of “signal to noise” ratio of NMR measurements: accumulation, apodisation (different multiplication functions), linear prediction, zero filling, effect of gyromagnetic factor (irradiated and detected nucleus), magnetic field strength, geometry factor, cryo and prodigy probe-heads, normal and special sample tubes, automatic sample changer, induced spin polarisation. Ultrashielded magnets. HPLC-NMR, sop and flow methods. Fast measurement techniques: Frydman experiment, Hadamard spectroscopy, NUS. Sructure elucidation strategies: combination of 2D and selective 1D NMR methods (NMR investigation of 20-hydroxy ecdysones), ultrafast bandselective 2D HSQC and HMBC measurements for differentiation of close signals.

4 hours

Mass spectrometry

Introduction: mass spectrometry basics, Ionization methods, Instrument types (analyzers)

Tandem mass spectrometry, hyphenated techniques (main focus on LC-MS): chromatogram types, limitations, compromises, trends in instrumentation, applications and methodology.

Biological mass spectrometry, proteomics. Selection of MS method. Examples.

8 hours

Chiroptical spectroscopy

e-CD spectroscopy: UV with linearly polarised light. Optical activity and chirality. Characteristics of linearly polarized light and its interaction with chiral systems, Cotton effect. Connectivity between CD, ORD and UV spectroscopy. Classification of chromophores: inherent chiral chromophores, coupling of achiral chromophores (exciton couplets), chirally perturbed achiral chromophores, e.g.: octant rule for carbonyl groups. Vibrational optical activity: v-CD and ROA (Raman Optical activity). Observation of Cotton effects in the frequency range of infrared and Raman vibrations. Ab initio calculation of v-CD spectra at DFT level for enantiomers’ structures.

4 hours

 X-ray diffraction

Basics of X-ray crystallography (crystal forms, elemental cell, asymmetric unit), introduction to theory of X-ray diffraction (Bragg's law, phase problem). X-ray diffraction (XRD) data collection techniques. Basics of structure determination from XRD data. Basics of protein crystallization, Interpretation of data from the Protein Data Bank

4 hours

ESR spectroscopy

EPR spectroscopy is a method to study paramagnetic compounds e.g. organic radicals and radical ions, triplet molecules, transition and rare earth metals. The basic concept of the measurement is the Electron-Zeeman interaction between the unpaired electron and an external magnetic field. An EPR signal is influenced by the extent of the magnetic field, by other magnetic nuclei around the unpaired electron and by the relaxation. This method can be used for the investigation of solutions, powders, single crystals of frozen solutions. In solution the fast motion of the molecules averages out the orientation dependent parameters however in the other three phases the main values of the tensors can be measured.

2 hours

 Lectures of students

Students will present 10-minute presentations about how they determined the structures of the unknown compounds using single- and two-dimensional NMR spectra. The lectures will be followed by a 5-minute discussion.

4 hours

ESR laboratory practice

The EPR instrument is shown in a laboratory demonstration, when the students can learn about the tuning and the adjustment of the measuring parameters (modulation frequency, magnetic field, number of measured points, MW power). During the practice the EPR spectra of a stable organic radical and a copper complex in solution and frozen solution is measured.

1 hour

 ESR calculation practice

During this practice the students calculate some EPR spectra from given magnetic nuclei and hyperfine coupling data. Than some printed EPR spectra is distributed to them and they have to determine the type of the magnetic nuclei and their hyperfine couplings.

1 hour

 X-Ray diffraction laboratory practice

Vapour diffusion crystallization practice, setting up a sitting drop plate by liquid handling robot and a hanging drop plate by hand. Imaging of the sitting drop plates by Formulatrix Imager, what is in the drop. Demonstration of an X-ray diffraction experiment on an Agilent, Supernova X-Ray diffractometer.

1 hour

 X-Ray diffraction practice in computer room

Introduction to practical use of the Protein Data Bank (www.rcsb.org), display of 3D protein structures by Pymol, analysis of ligand binding by PDB and Pymol utilities.

1 hours

 NMR practice for solving of NMR spectra

On the course of the practice the students (controlled by the instructor) evaluate single- and two-dimensional NMR spectra.

6 hours