Two-dimensional magnetic resonance spectroscopy

Under two-dimensional nuclear magnetic resonance ( 2D NMR ) refers to nuclear magnetic resonance spectroscopy method in which the intensities are recorded as a function of two frequency axes, that generates three-dimensional graphs. Two-dimensional NMR spectra provide more information about a molecule than one-dimensional NMR spectra and are therefore particularly useful in determining the structure of a molecule, especially for molecules whose structure is too complex to study with one-dimensional NMR.

description

The method is based on recording a series of spectra and during which the duration of a pulse parameter is continuously changed, whereby the phase and intensity of the spectra change systematically. By renewed Fourier transformation of the individual points of the spectra along this time axis, a two-dimensional one is obtained from the one-dimensional spectra.

If chemical shifts are plotted on one frequency axis and coupling constants on the other , we speak of two-dimensional J-resolved NMR spectra.

However, development did not stop at two dimensions. By varying other parameters, higher-dimensional spectra can be obtained. The required number of spectra and thus the measurement time increases exponentially. In the meantime, three-dimensional experiments (3D-NMR) with three frequency axes are almost routinely carried out, whereby the resonances of a different nucleus can be plotted on each axis, for example ¹ H, ¹³ C, ¹⁵ N or ³¹ P. However, it can also be on two axes ¹ H resonances and a hetero nucleus can only be removed on the third.

Two-dimensional nuclear magnetic resonance spectroscopy has several advantages over one-dimensional methods:

• By dividing it into two dimensions, you can also resolve complex spectra that can no longer be interpreted in one dimension due to strong overlaps.
• All the information about all groups of the molecule is obtained at once.
• You can select different physical interactions in two dimensions, according to which you want to separate. This enables various 2D-NMR methods.
• Furthermore, multiple quantum transitions, which are spin-forbidden to a first approximation, can be observed in two-dimensional NMR spectroscopy.

execution

The basic experiment of 2D-NMR can be divided schematically into four time segments: the preparation phase, the evolution phase, the mixing phase and the detection phase. During the detection phase, as in the conventional one-dimensional case, the signals are detected, digitized and stored at equidistant intervals Δt ₂ . In the preparation phase, longitudinal polarization is built up. It usually ends with a 90 ° pulse that generates transverse magnetization. During the evolution phase with the variable duration t ₁ , coherences develop under the influence of various factors (e.g. Larmor precession , spin-spin coupling ), which are coupled with each other in the mixing period, which is, however, not always necessary and converted into detectable transverse magnetization become. The evolution time t ₁ is gradually increased from experiment to experiment by a fixed value dt1. The FID (free induction decay) associated with each Δt ₁ value is stored separately. A two-dimensional matrix is ​​thus obtained which assigns a signal amplitude S (t ₁ , t ₂ ) to each pair (t ₁ , t ₂ ) . A two-dimensional Fourier transformation then converts the time signal S (t ₁ , t ₂ ) into the frequency signal S (ω ₁ , ω ₂ ).

${\displaystyle S(\omega _{1},\omega _{2})=\int \limits _{-\infty }^{\infty }\int \limits _{-\infty }^{\infty }S(t_{1},t_{2})e^{-i\omega t_{1}}e^{-i\omega t_{1}}dt_{2}dt_{1}}$

All FIDs are transformed as a function of t ₂ as usual . The resulting new data matrix now contains the NMR spectra arranged according to t ₁ in the rows (the ω ₂ direction). The next step is to transform again in the t ₁ direction. The representation takes place in the form of a 3D diagram (en: stacked plot) or as a contour line diagram in a square diagram, the axes of which represent the usual frequency scales.

history

According to a proposal by Jean Jeener from 1971, multi-pulse experiments with a systematically varied waiting time between two pulses were developed, which after Fourier transformation over two time ranges led to two-dimensional spectra. In the 1970s, Richard R. Ernst and his colleagues played a key role in the development of 2D-NMR. In 1991 he received the Nobel Prize in Chemistry. Through his work and that of R. Freemann, 2D-NMR became practicable. Kurt Wüthrich and others developed this 2D and multi-dimensional NMR into an important analysis technique in biochemistry, in particular for the structural analysis of biopolymers such as proteins . Wüthrich received the Nobel Prize in Chemistry in 2002 for this work.

Method of two-dimensional NMR spectroscopy

By selecting the spectra, for example through the type and selection of the time sequence of the pulses and calculation methods, different variants of 2D-NMR are possible. If you include all the variants, there are now several hundred different types of 2D NMR spectroscopy. For biochemical purposes, however, few play a role. In the most important processes in practice, two ¹ H or ¹ H and ¹³ C chemical shifts are linked to one another. The two-dimensional processes require a coupling of nuclear dipoles. This does not necessarily have to be a scalar coupling. As with the Kern-Overhauser effect , the interaction can also be of a dipolar nature, that is, it occurs through space. Frequently used procedures are:

COZY ( correlation spectroscopy )

Two-dimensional method in which nuclei of the same type ( ¹ H) (homonuclear COZY) or nuclei of different types (heteronuclear COZY) are correlated with one another via their scalar couplings. COZY spectra are symmetrical about the diagonals. With COZY, complex coupling patterns can be spatially rectified.

In the standard COZY, the preparation (p1) and the mixing periods (p2) each consist of a single 90 ° pulse, which is separated by the evolution time t1. The resonance signal from the sample is read over a period of t2 during the acquisition period.

¹ H COZY spectrum of progesterone

DOSY ( diffusion ordered spectroscopy )

Process in which, by means of field gradient NMR, molecules with different diffusion behavior can be recorded separately by NMR spectroscopy.

TOCSY ( total correlated spectroscopy )

Two-dimensional method in which nuclei of the same type ( ¹ H) are correlated with one another via their scalar couplings. Like COZY spectra, TOCSY spectra are symmetrical with respect to the diagonals. In addition to the signals detected in COZY, correlations appear in TOCSY between the starting nucleus and all nuclei that are indirectly connected to it via several couplings (spin system). The TOCSY experiment is particularly useful in determining the structure of high molecular weight substances with spatially limited spin systems, such as polysaccharides or peptides .

HSQC (English heteronuclear single quantum coherence )

Two-dimensional method in which chemical shifts of different scalar coupling nuclides are correlated. The HSQC spectra are often quite clear, since usually only signals from atoms that are directly bound to one another appear. Typical examples are ¹ H, ¹³ C and ¹ H, ¹⁵ N correlations.

¹ H– ¹⁵ N HSQC spectrum of a fragment of the protein NleG3-2.

HMBC ( heteronuclear multiple bond correlation )

Two-dimensional method in which chemical shifts of different scalar coupling nuclides are correlated. In contrast to the HSQC, the HMBC shows correlations across multiple bonds. ¹ H, ¹³ C correlations are particularly typical .

NOESY ( nuclear overhauser enhancement and exchange spectroscopy )

Two-dimensional method with which correlations are detected via the Kern Overhauser Effect (NOE) instead of via scalar couplings. With this method spatially adjacent cores can be recognized if they do not couple with each other in a scalar manner. There are both homo- and heteronuclear versions. This procedure is often used in structure elucidation.

Web links

Commons : Two-dimensional magnetic resonance spectroscopy  - collection of images, videos and audio files

Individual evidence

1. ↑ ^{a b} Matthias Otto: Analytical Chemistry . John Wiley & Sons, 2011, ISBN 3-527-32881-5 , pp. 270 ( limited preview in Google Book search).
2. ↑ ^{a b c} Horst Friebolin: One and two-dimensional NMR spectroscopy An introduction . John Wiley & Sons, 2013, ISBN 978-3-527-33492-6 , pp. 423 ( limited preview in Google Book search).
3. ↑ ^{a b c d e} Roland Winter, Frank Noll: Methods of Biophysical Chemistry . Springer-Verlag, 2013, ISBN 978-3-663-05794-9 , pp. 437 ( limited preview in Google Book search).
4. ↑ Eva-Maria Neher: From the ivory towers of science . Wallstein Verlag, 2005, ISBN 978-3-89244-989-8 , pp. 63 ( limited preview in Google Book search).
5. ^ Aue, WP, Bartholdi, E., Ernst, RR (1976): Two-dimensional spectroscopy. Application to nuclear magnetic resonance. In: Journal of Chemical Physics , 64: 2229-2246.
6. ^ Nobel lecture by Wüthrich (PDF; 399 kB).
7. ↑ Douglas A. Skoog, James J. Leary: Instrumental Analytics Basics - Devices - Applications . Springer-Verlag, 2013, ISBN 978-3-662-07916-4 , pp. 378 ( limited preview in Google Book search).