Sensitivity heteronuclear correlations

Information about the surface and interface structures in hexadecylamine-capped CdSe NC of 2 nm size has been obtained by a variety of 1H, 13C, 113Cd, and 77Se NMR techniques [342]. The 77Se CP-MAS-NMR spectrum showed five partially resolved peaks from surface or near-surface Se environments. It was possible to obtain 2D heteronuclear correlation (HETCOR) spectra between 1H and the other three nuclei despite the inherent sensitivity limitations (the 77Se- 3I-I HETCOR experiment required 504 h ). The latter experiment indicated that the methylene protons of the hexadecylamine chain interact with the surface Se atoms via a tilt of the chain toward the surface. The surface Se atoms were not seen to interact with thiophenol present, and it was suggested that thiophenol binds to a selenium vacancy at the surface. 

While the early days of LC-NMR and LC-NMR-MS were plagued by the poor sensitivity of the NMR spectrometer, the recent probe design advances have provided a means to potentially overcome this hurdle. As reported in the literature, it is possible to get both ID and 2D homo-nuclear and heteronuclear correlation data on sub micrograms of materials in quite complex mixtures utilizing cryogenic flow-probes in tandem with SPE peak trappings [98]. While these technologies are still in their infancy, they have the potential to revolutionize LC-NMR as a structure elucidation technique. 

When combining isotope filtering/editing with coherence transfer steps to multidimensional experiments, then further size restrictions apply. For example, isotope edited / filtered H TOCSY or COSY experiments are generally limited to systems of <10 kDa, because of their sensitivity to T2 relaxation. In larger systems, heteronuclear correlation spectroscopy can be used for the correspondingly labeled component, while structural information about both the labeled and unlabeled moiety can be extracted from isotope edi-ted/filtered NOESY spectra, respectively. 

The structures of the compounds were elucidated by a combination of NMR techniques (lH-, 13C-, and 13C-DEPT NMR) and chemical transformation, enzymatic degradation, and as well as mass spectrometry, which gives information on the saccharide sequence. A more recent approach consists of an extensive use of high-resolution 2D NMR techniques, such as homonuclear and heteronuclear correlated spectroscopy (DQF-COSY, HOHAHA, HSQC, HMBC) and NOE spectroscopy (NOESY, ROESY), which now play the most important role in the structural elucidation of intact glycosides. These techniques are very sensitive and non destructive and allow easy recovery of the intact compounds for subsequent biological testing. 

Two classes of heteronuclear correlation experiments are used to provide information about one-bond and two/three-bond carbon-hydrogen attachments. These experiments are 100-fold less sensitive, as they require detection of signals from 1% of the molecules containing The earlier experiments such as... 

HETCOR and COLOC involve C detection these have been superseded by more sensitive Undetected HMQC, HSQC, and HMBC experiments, which provide ca. 30-fold sensitivity improvement over C detection. Heteronuclear correlation experiments provide simpler spectra (a single peak is observed for each C-H attachment) and they take advantage of the much greater C spectral dispersion. 

Despite these impressive sensitivity gains when directly observing the X-spin, the more modem approach is to observe the X-spin indirectly through the coupled proton when possible, which can be achieved through a number of heteronuclear correlation experiments. As these methods additionally employ proton observation, they benefit from a further theoretical gain of (yhigh/Viow) over X-observe schemes (see Eq. 4.2). These topics are pursued in Chapter 6, and should be considered as potentially faster routes to X-nucleus data. 

Figure 6.8. A comparison of signal suppression methods used in proton-detected heteronuclear correlation experiments (see descriptions in text). Spectrum (a) is taken from a conventional ID proton spectrum without suppression of the parent resonance and displays the required satellites. Other spectra are recorded with (b) phase-cycling, (c) optimised BIRD presaturation, and (d) pulsed field gradients to remove the parent line. All spectra were recorded under otherwise identical acquisition conditions and result from two transients. Complete suppression can be achieved with gradient selection, but at some cost in sensitivity in this case (see text).

This section focuses on the coupling evolution that occurs during the pulse sequences of heteronuclear correlation experiments. Because polarization transfer is a requirement of intensity optimized heteronuclear correlation spectroscopy this section also acts as a short introduction to coherence transfer by polarization transfer. A coherence transfer from a sensitive nucleus, e.g. in most experiments, to a nucleus contributes a gain of signal intensity. To obtain the required antiphase coherence for the transfer step the coherence must evolve exclusively by heteronuclear coupling during a free precession periods. This can only be assumed in theoretical spin systems. In real samples three aspects make this ideal approach more difficult ... 

Comparative sensitivity gain by coherence transfer in heteronuclear correlation spectroscopy [5.3]. 

Heteronuclear correlation experiments with IR detection in the direct acquisition period t2 are often called inverse detected experiments. The term "inverse" is also used to describe probeheads that are constructed with the coil as the inner coil and the rf coil for the heteronuclear frequency as the outer coil. The outer coil has a lower sensitivity because of the lower fill factor. This term may also be used to distinguish between two classes of experiments. The HETCOR and COLOC experiments belong to the class of direct detection experiments with take advantage of the coherence transfer from the sensitive nucleus to the relatively insensitive heteronucleus. This type of experiment is illustrated by the first entry in Table 5.22. However experiments in this category are no longer popular and have been superseded by inverse detected experiments, shown in the second entry in Table 5.22. In a comparison of the different types of experiments the IH detected heteronuclear correlation experiments have three distinct advantages over the detected experiment ... 

In the early days of heteronuclear correlation spectroscopy, a gradient free version of the double low-pass filter was proposed [5.212, 5.213], but the necessity to suppress unwanted coherences, particularly in INADEQUATE experiments, has forced the design of a gradient version [5.214]. The application of this filter is not just restricted to INADEQUATE experiments it has also become the element of choice in ACCORD-HMBC and ACCORD-CIGAR-HMBC experiments to reduce residual one-bond correlations. These experiments are very sensitive to the suppression of one-bond... 

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