Cryo-probes

Fig. 14.2 Population of the bound state of a complex as a function of the (molar) concentration of the unobserved substance for different KD values. The concentrations of the observed substance are 100 pM (cryo probe, panel A) and ImM (standard equipment, panel B), corresponding to typical experimental situations. The KD areas shaded in...

Fig. 14.2 displays the fraction of bound ligand as a function of the dissociation constant for two different ligand concentrations (A) 100 pM, about the detection limit for ID experiments by use of cryo-probes, and (B) 1 mM, a concentration for standard equipment. At these concentrations, typical measurement times are in the range of several minutes. From the figure the following conclusion can be drawn for ligand-observe techniques ... 

Under the assumption that a given NMR method (see below) allows for the detection of 10% bound ligand, the upper limit of the KD that will cause appreciable differences in the spectra equals the concentration of the molecule under observation (e.g. for a cryo-probe at a typical [substance] [protein] ratio of 10 1, KLl values below 100 mM can be detected). 

So for very limited sample amounts, it usually is best to go to the highest field available, with the smallest diameter cryo-probe possible. A cryo-capillary flow microprobe that accepts a few nanograms of material in approximately 1 pi of solvent yields the highest sensitivity for mass limited samples. 

We notice that the S/N grows proportionately to Vns, where ns is the number of scans or repetitions of the pulse program. This relationship is not typically a problem with H experiments where only a few pg to a mg of material is enough to get good S/N in a few scans. As mention previously, l3C is 6000 times less sensitive than H, and therefore requires either more sample (N), higher field strengths B0 (or better probe technology, i.e., a cryo-probe), or an increase in the number of scans (ns). 

The chemical structure of natural products can be identified quickly with a limited amount of materials by utilizing NMR equipment containing cryo probes [60]. More predictable chemical shifts coupled with a reasonable amount of published and internal NMR data [61] will significantly improve the time and accuracy of the structure elucidation process [62,63,64],... 

Protein concentrations in solution are often limited by solubility and aggregation (and, of course, protein availability) to a maximum of 0.5-5 mM. Today most NMR structural studies are performed at 1-2 mM protein concentrations, although it can be expected that new technologies leading to increased sensitivity in NMR experiments (increased field strength, improved probe design, cryo-probes etc.) will lower the concentration requirements to the 100-mM range within the next few years. 

Figure 1 NMR spectra of tritiated o-methoxyacetophenone (o-MeO-C6H4COCH2T, 68pCi) observed (A) at 320.13MHz with a 5 mm duai-proton/tritium probe, (B) at 533.5 MHz with a 5 mm ieak-proof seiective excitation proton/tritium probe, (C) at 533.5 MHz with the 5 mm seiective excitation proton/tritium cryoprobe, and (D) the same compound at iower activity (11 pCi) using the 5 mm seiective excitation proton/tritium cryoprobe at 533.5 MHz. (Reproduced with permission from Bioxsidge JP, Garman RN, Giiiies DG, Jones JR, and Lu SY (2004) Deveiopment and appiication of a tritium cryo-probe H NMR studies at the microcurie (megabecquerei) ievei of radioactivity, in Dean DC, FiierCN, and McCarthy KE (eds.) Synthesis and Application of Isotopically Labelled Compounds, voi. 8. Chichester John Wiiey Wiiey.)...

In the short time interval (5 years) since cryo-probes were commercialized they have become an indispensable accessory for NMR. Initially, they were available on 500 MHz instruments but now they have been installed on 700 and 800 MHz spectrometers. The more recent cryostats, thanks to ingenious design, are now less sensitive to disturbances such as changes in room temperature and helium evaporation rates. All of these factors will ensure considerable improvements in the performance of these ultrastabilized magnet systems. 

Fig. 7.2.4. Mean of temperature scores (j-axis, left) measured at the tip of the cryo probe and mean diameters of the ice-ball (y-axis, right) at each minute (x-axis) during the procedure in the first 16 patients...

---