2D spectroscopy

For a manufacturer the difficulty is to estimate future developments or trends in NMR microscopy. Based on dedicated laboratory-made hardware developed by the NMR microscopy users and on their requests for new commercial hardware and software, the following topics could become more important micro-coil applications, multiple receiver systems and multi-coil arrangements, NMR microscopy at very high magnetic fields, MAS imaging and localized 1H MAS spectroscopy and localized single-shot 2D spectroscopy. There are no clear-cut distinctions between most of the individual topics, as will be discussed in the following sections. 

The separation of interactions by 2D spectroscopy can be compared with 2D chromatography. In a onedimensional thin layer or paper chromatogram, the separation of the constituents by elution with a given solvent is often incomplete. Elution with a second solvent in a perpendicular direction may then achieve full separation. In NMR spectroscopy, the choice of two solvents is replaced by the choice of two suitable (effective) Hamiltonians for the evolution and detection periods which allow unique characterisation of each line. 

Figure 4.1 Schematic diagram of a general-purpose CSTR recycle system for in situ investigations of liquid-phase homogeneous catalyzed reactions. The blocks represent in-line instruments and their signals, namely, (i) sets of scalar valued measurements (sensors), (ii) sets of vector valued measurements (ID spectroscopies) and (iii) sets of matrix valued measurements (2D spectroscopies). The recycle time for the system is given by t.

The acronym COSY stands for Correlated SpectroscopV and this technique is widely used to determine all of the coupling interactions in a single experiment. This proves to be more efficient than the decoupling experiment in which each signal is irradiated in turn to determine its coupling partners. COSY involves a multiple pulse sequence (which we do not need to know anything about in order to use the technique) and is an example of two-dimensional (2D) spectroscopy. 

Lanthanides have been used as substitutes for calcium in calcium binding proteins since the early days in NMR [155,156]. Remarkable information was obtained on systems such as Yb(III)-substituted parvalbumin (which contains a typical calcium binding site called EF-hand [157,158]) from ID spectroscopy alone [159-164], later complemented by 2D spectroscopy [165]. More recently, pseudocontact shifts and longitudinal proton relaxation times have been used to... 

The nuclear Overhauser effect is something very dear to senior NMR researchers. It is the effect which allows us to know which magnetic nuclei are close to other magnetic nuclei, and information on their distances becomes available. Its understanding used to be a must to move towards 2D spectroscopy. Now times are somewhat changed, but... 

Two-dimensional (2D) spectroscopy is used to obtain some kind of correlation between two nuclear spins 7 and J, for instance through scalar or dipolar connectivities, or to improve resolution in crowded regions of spectra. The parameters to obtain 2D spectra are nowadays well optimized for paramagnetic molecules, and useful information is obtained as long as the conditions dictated by the correlation time for the electron-nucleus interaction are not too severe. Sometimes care has to be taken to avoid that the fast return to thermal equilibrium of nuclei wipes out the effects of the intemuclear interactions that are sought through 2D spectroscopy. 

Of course, these considerations regarding the folded spectra and decreased dynamic range of the ADC hold for any kind of 2D spectroscopy. We have mentioned them here because such problems happen to have been afforded in the NMR literature on paramagnetic compounds in connection with NOESY spectra. 

An obvious extension to 3D spectroscopy from 2D spectroscopy is the homonu-clear NOESY-NOESY [34]. There are two t variable times and one tj, which after Fourier transform provide three frequency domains. The 3D NOESY-NOESY spectrum of met-myoglobin cyanide, which contains low spin iron(III) in a heme moiety (see Fig. 5.7), has been successfully measured [35]. In Fig. 8.22 a slice of the 3D spectrum is shown at the I2-CH3 height. On the diagonal it shows all the dipolar connectivities between I2-CH3 and other protons off-diagonal... 

In principle, all the combinations of homonuclear 2D spectroscopies can be performed to originate a 3D spectrum (COSY-COSY, NOESY-COSY, NOESY-TOCSY, etc.). The considerations made in this chapter for the most basic experiments can be easily extended to their combinations. The general guideline should always be that the more complex the pulse sequence is, the more the experimental sensitivity will suffer from fast nuclear relaxation. 

Weft or Superweft sequences are also widely used in 2D spectroscopies (see Section 9.4) in general, it is sufficient to place the 180°-t part of the sequence in front of any 2D sequence to achieve the desired signal suppression. 

This is where two-dimensional (2D) spectroscopy comes into play, for example, by correlating one dimension where the anisotropic interactions are preserved with a (high-resolution) dimension where they are removed. That is, both objectives can be achieved within one two-dimensional experiment. Like conventional one-dimensional (ID) NMR techniques, 2D techniques were first applied to liquid or solutions, where they provided invaluable information for the structure assignments in biomolecules [7]. Later, after the... 

Two-dimensional (2D) spectroscopy The silicon vidicon is a two-dimensional OID with an area-array target comprising a few hundred thousands dicrete photodiodes. Since these diodes can be randomly read out by the scanning beam, the detector is capable of performing some very useful spectrometric tasks ... 

We are beginning to develop a detailed understanding of these methods (18,21,30,33,34,37-40,42,44,47-49), many of which are described in this book. We have recently demonstrated a series of novel nonlinear all-IR spectroscopic techniques (IR-pump-IR-probe, IR-three-pulse photon echoes, IR-dynamic hole burning, IR-2D spectroscopy), all of them utilizing intense femtosecond IR pulses, with the intention to develop new multidimensional spectroscopic tools to study the structure and the dynamics of proteins (30,31,41,42,50-53). We shall summarize in this contribution our work, its underlying principles, and its applications. 

Multidimentional nonlinear infrared spectroscopy is used for identification of dynamic structures in liquids and conformational dynamics of molecules, peptides and, in principle, small proteins in solution (Asplund et al., 2000 and references herein). This spectroscopy incorporates the ability to control the responses of particular vibrational transitions depending on their couplings to one another. Two and three-pulse IR photon echo techniques were used to eliminate the inhomogeneous broadening in the IR spectrum. In the third-order IR echo methods, three phase-locked IR pulses with wave vectors kb k2, and k3 are focused on the sample at time intervals. The IR photon echo eventually emitted and the complex 2D IR spectrum is obtained with the use of Fourier transformation. The method was applied to the examination of vibrational properties of N-methyl acetamid and a dipeptide, acyl-proline-NH2.in D20. The 2D IR spectrum showed peaks at 1,610 and 1, 670 cm 1, the two frequencies ofthe acyl-proline dipeptide. Geometry and time-ordering of the incoming pulse sequence in fifth-order 2D spectroscopy is shown in Fig. 1.3. 

Figure 1..3. Geometry and time-ordering of the incoming pulse sequence in fifth-order 2D spectroscopy. Aki c ki-k,% Ak2 = k2-k2 . Only one of the nine possible signal pulses in the direction ks= Aki + kp is shown. Inset shows the elementary Raman process (Mukamel et al., 1999). Reproduced with permission.

In 2D spectroscopy the Gaussian function is often preferred over exponential multiplication because little broadening at the base of the resonance is induced by this filter, and significant sensitivity improvement can still be obtained. 

Barjat, H., Morris, G.A., Smart, S., Swanson, A.G. and Williams, S.C.R. (1995) High-resolution diffusion-ordered 2D spectroscopy (HR-DOSY) - a new tool for the analysis of complex mixtures. J.Magn. Reson. Ser. B, 108, 170-172. 

Volume selection is particularly useful for spatially localized 2D spectroscopy, because acquisition times for 2D spectroscopic imaging would be prohibitively long. Localized 2D spectroscopy is of interest, for instance, to detect information hidden beneath overlapping signals in ID spectra [Alol, Berl, Cohl, Sotl] as well as to follow reaction kinetics [Ball]. 

Fig. 9.1.9 [Blal ] Timing diagrams for rf and gradients applicable to spatially resolved 2D spectroscopy, (a) Spatially localized COSY, (b) Spatially localized NOESY.

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