Probes cryogenic

With the use of cryogenic probes, carbon detection becomes possible in protein NMR experiments [23]. This may be required for perdeuterated proteins or when signal loss in INEPT transfer steps becomes prohibitively large. 

For mass-limited samples, small-diameter (conventional) probes present an attractive alternative to cryogenic probes. In collaboration with Hoffmann-La Roche, Bruker has recently developed a 1 mm probe with a sample volume of 5 pL. This probe offers a mass sensitivity that typically is fourfold higher than a conventional 5 mm probe. An example displaying a [13C, 1H]-HSQC of 1 pg Ibuprofen at 13C natural abundance is shown in Fig. 3.6 below [25]. 

Kd values one order of magnitude lower than the concentration of the observed substance cannot be discriminated by their effect on the spectra at the 10 1 sub-stance protein ratio (e. g. for a cryogenic-probe a 10 pM ligand will cause the same spectral differences as a 10 nM ligand, provided that both are in fast exchange). 

Figure 6.48 Two-dimensional NOESY spectrum (mixing time = 2 s) of the ether glucuronide of 3-methoxy-paracetamol at 600 MHz in a 3-mm cryogenic probe head (total experiment time 20 h). The sample was recovered from a conventional 3-mm LC probe head after a triple trapping SPE-NMR run (result shown in Eigure 6.41). Reproduced from [59] with permission from Elsevier.

Furthermore, in a pharmaceutical development environment (working to GMP), redeveloping a method is not a simple task since peak tracking (by LC-MS) and validation may be required and, in the case of registered methods, results obtained by modified methods may not be acceptable. In such cases, it may be more efficient to use a preparative approach followed by conventional tube NMR where optimum sensitivity can be obtained through the use of cryogenic probes (Figure 6.48) or low-volume 1 mm probes [90]. 

Keun, H.C. et al.. Cryogenic probe 13C NMR spectroscopy of urine for metabonomic studies, Anal Chem., 74, 4588, 2002. 

Kim, Y. W., Earl, W. L., and Norberg, R. E. (1995). Cryogenic probe with low-loss transmissionline for nuclear-magnetic-resonance./. Magn. Reson. Ser. A 116,139-144. 

Voehler, M. W., Collier, G., Young, J. K., Stone, M. R, and Germann, M. W. (2006). Performance of cryogenic probes as a function of ionic strength and sample tube geometry. J. Magn. Reson. 183,102-109. 

Griffin, in a review of metabonomics, also notes the impact of cryogenic probe capabilities.252... 

A second natural product study reported in 2003 that used the sensitivity of cryogenic probe capabilities and the HSQC-TOCSY experiment to advantage was the report by Preiss et al.25S that detailed the elucidation of the structure of two new asterosaponins from the starfish Asterias rubens. The structure of one of the molecules characterized (110) of which only 0.3 mg was isolated, is shown. 

Moskau and Zerbe262 in a general review of cryoprobe hardware and performance also detailed ways of quickly recognizing component malfunction in cryogenic probe systems, which from the author s own experience is an area from which others might benefit. 

Sensitivity can furthermore be increased up to fourfold by using cryogenic probe technology, in which the radio frequency (rf) transmitter and preamplifier coils are super-cooled to 20K by helium gas that ensures higher signal-to-noise of the electrical signal. Vacuum insulation around the rf coil allows the NMR sample situated only millimeters away from the coil to be measured at ambient temperature. 

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