*2.1. Liquid State*

As hydrogen bonding is the subject, the most relevant protons to exchange are those of OH, NH or SH groups. Deuteration can easily be achieved either by shaking with D2O in, e.g., chloroform or methylene chloride and subsequent drying with water-free sodium sulphate or by dissolution in CH3OD followed by evaporation. In case of exchange of CH protons this depends very much on the compound. An example is imidazolium acetate liquid ionic liquids which have been used to catalyze H-D exchange in 2-alkanones using CD3OD as deuterium source. Long-range isotope effect on 13C chemical shifts is measured [7]. For a different case see Section 3.1. Introduction of 18O may be obtained by exchange, whereas 15N usually requires a synthetic scheme. In the case of deuteration, a degree of deuteration of ~70% is desirable as some lines in the spectrum may become broad due to couplings to deuterium and a clear-cut difference in intensities between molecules with H and those with D is required in order to determine the signs of the isotope effects. As slow exchange is a prerequisite to measure isotope effects directly, low temperature may be necessary. For examples of very low temperature, see references in this paper and previous papers by the Limbach-Tolstoy group [6]. Cooling down may not only slow down the XD exchange but may also lead to observation of isotope effects of single rotamers as seen in, e.g., 2,6-dihydroxyacylaromatics (see Section 3.5). Exchange can be slowed down by use of a hydrogen bonding solvent such as DMSO [8]. In the case of ammonium ions, exchange may be slowed down by acidification.

For compounds only soluble in water, the deuterium isotope effects can be determined by recording spectra with varying ratios of H2O/D2O followed by extrapolations to 0 and 100% D2O.

Isotope effects can in most cases be determined by simple 1D NMR spectra. However, for <sup>1</sup>ΔN(D) isotope effects it is an advantage to use indirect techniques. D.F. Hansen et al. have developed a 13C-detected 15N double-quantum NMR experiment [9]. In cases with exchange, the HISQC technique is useful [10].

Assignment of isotope effects in molecules with more than one site of e.g., deuteration can be achieved by utilizing different degrees of incorporation or different rates of incorporation. Examples are in β-diketones and in enaminones. In these cases, the hydrogen at the central carbon exchange much more slowly than the XH protons. In other cases, either a spatial separation or a comparison with similar compounds with fewer sites of deuteration can be used (see Figure 1)

**Figure 1.** Deuterium isotope effects on 13C chemical shifts. Values in brackets are due to the deuterium in brackets. Data from Ref. [11].
