NMR-TUTORIAL GUIDE_2.0

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2D 1H,13C HSQC: INTRODUCTORY REMARKS

The idea, the basic principle and the advantages of 2D- (or multi-) dimensional NMR correlation spectroscopy have been discussed already in the previous chapter.
For the 2D 1H,1H COSY experiment it has been demonstrated that by extending signal sampling to two dimensions of time t1, t2 finally and after two-fold Fourier transformation a 2D spectrum with two frequency domains F1, F2 can be generated.

This of course also applies for the 2D 1H,13C HSQC experiment :
General Structure 2D NMR Experiments
As already outlined in The Tutorials Elements 2D spectra are characterized by two frequency axis F1, F2.

Hence and in contrast to 1D experiments with the signal intensity recorded along one time-axis t (the acquisition time) the signal intensity is measured as a function of two time-axis t1 and t2 respectively.

This is accomplished by a series of sub-experiments with one (or more) delays incremented in a second time domain t1 from sub-experiment to sub-experiment and with the signal intensity acquired for each sub-experiment along a single time axis t2 (the acquisition time).
Any 2D experiment can be subdivided into four periods, the preparation period P, the evolution period E, the mixing period M and the detection period D respectively:
P
The preparation period P allows the investigated spin system in a running 2D experiment to relax and to reach a (pseudo-) equilibrium state.
Adequate polarization for the subsequent scan optimized with respect the overall signal-to-noise ratio is built up by longitudinal relaxation and residual transverse magnetization potentially affecting the signal of subsequent scans in an uncontrolled manner is allowed to disappear by transverse relaxation.
E
The evolution period E the length t1 of which is incremented from sub-experiment to sub-experiment allows the spin-system to evolve under the influence of a predefined Hamiltonian H1.
For the 2D 1H,1H COSY experiment and as an example the spin system evolves under the influence of proton chemical shifts, homonuclear (and if present heteronuclear) couplings and relaxation.
M
In the mixing period M a component of magnetization, say of spin i, present at the end of the evolution period t1, is transferred through some mixing process to another spin j.
The size of the transferred component varies as function of t1, hence it is said to be modulated in t1. This modulation detected for spin j reflects the “history” of spin i in the evolution period t1, to which it is connected by some kind of interaction or coupling.
For the 2D 1H,1H COSY experiment this interaction is the indirect (scalar) homonuclear coupling nJHH effective between protons i and j.
D
The final detection period D allows the signal of nuclei to be detected as a function of t2 under the influence of a Hamiltonian H2. 
Whereas the signals are modulated in t2 under the influence of H2, the intensities (and/or phases) of the signals are additionally modulated for the individual spectra acquired with incremented t1, hence are modulated under the influence of H1. 
For the 2D 1H,1H COSY experiment the signal of a spin j is modulated in t2 as a function of its chemical shift δj and its couplings nJjk and is additionally modulated in t1 as a function again of of δj and its couplings nJji, but and most importantly also under the influence of the chemical shift of any indirectly coupled spin(s) δi and its couplings nJik.

It is this latter modulation which has made 2D NMR experiments such popular and which allows various kinds of coupling interactions among different spins to be visualized most clearly in 2D spectra and to exploit corresponding (shift-) correlations for unraveling spin-spin networks and hence molecular structures.
PROCESSING OF 2D NMR DATA
Performing 2D experiments has a number of 2D specific consequences both in the setup the experiment and in the processing the acquired raw data.
Whereas the set-up of 2D experiment will be discussed in the subsequent sections the most important facts and consequences resulting from two-dimensional data acquisition will be examined here:
  • processing in general depends on the 2D experiment type but also on the sample under investigation and the sighted spectral information, i.e. processing is more resolution or sensitivity driven.
  • as already addressed in the introductory part The Tutorials Elements 2D experiments in general yield a two-dimensional data matrix with the signal intensity, phase or both these properties depending on two time-axis t1 and t2.
  • consequently, two Fourier Transformations with respect to t1 and t2 have to be performed with this 2D raw data for obtaining the final 2D spectrum with frequency axis F1 and F2 respectively.
  • furthermore, and analogous to 1D data processing additional processing in the two time- and the two frequency domains may and has to be performed such as e.g. weighting (apodization) and phasing respectively.
  • 2D processing is performed stepwise and starts typically with the time-domain processing of the individual t2-FIDs (acquired for a number of t1 values) completed by the first Fourier-Transformation across t2:
  • before this first FT the time-domain signals, i.e. the t2-FIDs, are processed in the same way as 1D FIDs.
  • appropriate weighting functions and corresponding parameters are chosen according to the given experiment and according to the wanted information.
  • as with 1D data artificial sensitivity enhancement is envisaged with inherently low sensitive experiments, such as 13C,1H or 15N,1H heteronuclear HSQC.  Artificial resolution enhancement – assuming sufficient digital resolution – on the other hand is applied with pure, highly sensitive 1H 2D data, such as the 2D 1H,1H COSY experiment for resolving the fine structure of cross-peaks and hence for determining coupling constants of adequate precision.
  • after the first FT with respect to t2 the 2D data matrix is transposed. Corresponding data points of the individual F2-spectra are used to build up FIDs in t1, i.e. the kth point of each F2 spectrum is used to construct the kth FID in t1. This procedure is repeated for all data points of the F2 spectra.
  • after transposition a second time-domain processing before the second FT, both now with respect to t1 are performed before final processing in both frequency domains.
  • for t1-processing adequate weighting considering both, the needs for either highest sensitivity or highest resolution on the one side, but also the t1-FIDs “natural”, 2D experiment specific shape on the other side, is probably the most important step. Additionally, and most popular with insensitive 2D experiments more sophisticated procedures for artificial sensitivity enhancement such as linear prediction or maximum entropy methods (MEM) may be applied at this stage.
  • after the second FT final processing in both frequency domains include adequate baseline correction, phasing, calibration and less common steps such as 2D signal integration, spectral symmetrizing or tilting.
  • as a result, there is not a single processing scheme for all types of 2D NMR data but the individual processing steps and corresponding processing parameters are highly experiment specific as demonstrated in the subsequent sections and chapters and various approaches with carefully selected processing steps and parameters exist instead.
THE BENEFITS OF 2D NMR SPECTROSCOPY
Compared to a 1D spectrum improved spectral resolution and clarity is achieved with a 2D spectrum. Spreading a spectrum into two frequency dimensions allows the presence (and absence) of mutual J-coupling to be visualized most clearly and the coupling network of the investigated compound to be disentangled most conveniently. Thereby the signals of the individual protons are correlated taking advantage of the proton specific chemical shifts.
 
As outlined in the introductory part of the last chapter proton signals may be correlated not only according to scalar J-coupling but also according to other intra- and intermolecular spin-spin interactions. Dipolar J-coupling among closely spaced protons giving rise to NOE effects or chemical/dynamical exchange among protons yield additional valuable structural information and may best be investigated by correspondingly designed 2D experiments.
        
HETERONUCLEAR 2D NMR SPECTROSCOPY IN GENERAL
However, 2D NMR spectroscopy is not restricted to experiments for probing mutual interactions among protons. It can also be applied in a homonuclear manner to other nuclei, e.g. fluorine (2D 19F,19F-COSY), phosphorous (2D 31P,31P-COSY) and even carbon (2D 13C, 13C-COSY).

Furthermore, and most popular 2D NMR spectroscopy may also be performed in a heteronuclear manner correlating the signals of different types of J-coupled nuclei such as protons and carbons or protons and nitrogen to unravel the corresponding coupling networks of the investigated compound.

Before concentrating on 1H, 13C shift-correlation and addressing the 2D 1H,13C HSQC experiment in more detail a few comments concerning heteronuclear 2D NMR spectroscopy in general will be given:
SENSITIVITY
  • with low-abundant nuclei such as 13C or particularly 15N the inherent low sensitivity associated with long measuring times is in the focus. Therefore, the second nuclei of corresponding heteronuclear 2D experiments is typically the proton taking advantage of its high sensitivity.
  • two main types of 2D 1H, 13C 2D experiments exist with respect to the number of chemical bonds connecting the coupled nuclei. This chapter focuses primarily on 2D experiments correlating 1H and 13C signals of nuclei coupled selectively across one (1JCH) chemical bond will be investigated. The sub-chapter Variants of the 2D HSQC sequence and related experiments addresses an experiment and a few of its variants dedicated to selectively detect 1H, 13C-J-coupling across two, three or more (nJCH) chemical bonds.
  • an “arsenal” of considerable extent with different variants for 1JCH- (and nJCH-) correlated 1H, 13C 2D experiments are in use. They are designed for achieving at the same time highest sensitivity, best performance/robustness and for trapping additional valuable spectroscopic information facilitating signal assignment and structure elucidation in general.
  • highest sensitivity is obtained with so-called 1H-detected variants. Starting from protons transverse carbon coherence enhanced by the first polarization transfer (1H ⇨ 13C) is generated, evolves in the first time domain t1 and is back-transferred to protons (13C ⇨ 1H) before final signal detection in t2. The higher sensitivity of protons results for two reasons, exploiting the proton’s higher gyromagnetic factor in the initial polarization transfer and exploiting the protons much higher resonance frequency accompanied with a higher rf-detection sensitivity in final signal sampling.
  • a further significant increase in sensitivity can be achieved by using cooled probe heads (cryo probes). Cryo probes using helium- or nitrogen cooled RF coils and preamplifiers are available to deliver a sensitivity enhancement over room temperature (RT) probes. Cryo probes with the innermost coil assigned either to 1H (for highest 1H-sensitivity) or to 13C (for highest 13C-sensitivity) allow 1H, 13C 2D experiments to be performed with final 1H- or 13C detection respectively.
    
  • last but not least, in terms of sensitivity, special detection methods such as non-uniform (instead of conventional uniform) sampling, or  e.g. the ASAP (Acceleration by Sharing Adjacent Polarization) procedure, both of which shortens measuring times, are used with 2D 1H, 13C experiments.
THE EXPERIMENT'S PERFORMANCE AND ROBUSTNESS
  • to improve the experiment’s performance and robustness various accessories with magnetic field gradients and shaped pulses as the most important are used today by default. Magnetic field gradients allow the wanted coherence pathways to be selected highly selectively, an indispensable prerequisite for suppressing the unwanted huge 1H signal of the all-12C isotopomers with 2D 1H,13C experiments with final 1H detection. Shaped pulses allow the broadband-capability and concomitantly the overall sensitivity to be enhanced in general. They are most important for the 13C-channel and with spectrometers operated at high magnetic field strengths.
ADDITIONAL SPECTROSCOPIC INFORMATION
  • variants of the basic 2D 1H,13C experiments exist which - besides correlating proton and carbon signals - allow to extract additional valuable spectroscopic information. The individual multiplicities (CH, CH2, CH3) of the carbons involved may be visualized or the cross-polarization (TOCSY-) behavior of the individual protons may be detected most conveniently, i.e. resolved in the first (13C-) frequency dimension thus free from annoying superpositions.
ADDITIONAL
  • in contrast to the symmetric homonuclear 2D spectra with a diagonal and with cross- and diagonal peaks, heteronuclear 2D spectra are most obviously non-symmetric and show only cross-peaks.
  • for the sake of completeness and to focus on a more technical aspect, the different methods for achieving quadrature detection in both time domains should be pointed out. As in homonuclear 2D experiments, these are used to display the heteronuclear 2D spectra either in magnitude or (preferably) in phased mode.
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