Probeheads

The signal-to-noise ratio of a signal correlates to the ratio of induced voltage to noise voltage [1, 20]. It can be described as 

Although ubiquitin displays very favorable relaxation properties, these spectra indicate the potential for performing these experiments at 13C natural abundance for smaller proteins. 

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]. 

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H-NMR studies were performed on a Bruker MSL-400 spectrometer operating in the Fourier transform mode, using a static multinuclei probehead operating at 400.13 MEtz. A pulse length of 1 iis is used for the 90° flip angle and the repetition time used (1 second) is longer than five times Tjz ( H) of the analyzed samples. 

In practice it is usually unnecessary to determine exact pulse widths for each sample we can use approximate values determined for each probe-head, except in certain 2D experiments in which the accuracy of pulse widths employed is critical for a successful outcome. Proper tuning of the probehead is advisable, since pulse widths will normally not vary beyond 10% with well-tuned probeheads. 

Further expansion of the probehead to more coils is also possible, with a concomitant reduction in the total amount of material needed for a kinetic study. 

Table 7.70 lists some of the main features of LC-NMR hyphenation. At 11.7T (500 MHz for 1H) a 4 mm LC-NMR flow-probehead readily provides a detection limit of ca. 5 p,g for on-flow (lmLmin-1) and 150ng for stopped-flow (in 3 h) for a typical 350-Da substance. Miniaturisation and hyphenation of NMR to various capillary-based microanalytical systems (LC, CZE) was described [650]. 

A long capillary with a computer-controlled switching valve (the instruments must be separated by 2-3 metres because of the strong magnetic field) connects the exit from the HPLC with the probehead. The latter is completely different in its construction from conventional probeheads instead of the NMR tube there is a small flow cell, the volume of which is 40-100 pi. The transmitter and receiver coils are attached directly to the cell in order to maximize the sensitivity. 

The next step is to set the same conditions for the HPLC system which is coupled with the NMR spectrometer. The field homogeneity of the probehead is first optimized (shimmed) using the same separation column and solvent mixture. 

Over 20 spin-Vi nuclei are available to the NMR spectroscopist. Most are very insensitive with respect to the proton or even to carbon-13, but modern NMR techniques still make almost all of them easy to study. A few have NMR resonance frequencies which are very low, and cannot be measured using standard probeheads. 

Fluorine-19, like phosphorus-31, is a spin-Vi nucleus with 100% natural abundance. The signals it produces are almost as strong as those of the proton, and the resonance frequency at a given field is also relatively close to that of the proton. Although for many years it was in fact necessary to have a special probehead for fluorine-19, those days have gone and fluorine has become a completely normal nucleus. 

In 2000, Gan proposed the previously mentioned STMAS method, which is similar to MQMAS in that it can be performed on a standard MAS probehead, but uses a different coherence pathway [141]. The STMAS protocol, schematically described in Fig. 10, relies on excitation of the SQ ST coherences of order q (with q yk 0 and p = —1), instead of symmetric MQ coherences with q = 0. Note that such excitation inevitably involves the CT, as well. The ST coherences are converted to the CT coherence at t = r/(l + k) using suitable rf-pulse pulse(s). 

In order to reveal sources of artifacts and noise we will give a brief description of spectrometer hardware and probehead technology. Of course, spectrometer manufacturers do their best to construct hardware with optimal performance. However, experience shows that all hardware components may occasionally fail. It is one of the goals of this chapter to present ways to recognize malfunction quickly and to locate the source of problem. 

The most commonly used probehead is the inverse dual probehead, which contains an additional coaxial coil that is tuned to resonance frequency, surrounding the detection coil for hetero-... 

The hardware required for PEG work, although commercially available, is not usually included in the basic version of spectrometers. It consists of a gradient accessory, a gradient amplifier, a shielded probehead equipped with a z-gradient coil and a pulse shaper (which in itself is an integral part of the gradient accessory). 

As is the case in most gradient-enhanced pulse sequences, GROESY spectra should preferably be obtained with non-spinning samples. In our spectrometer, a Bruker ARX-400 equipped with an inverse broadband probehead incorporating a shielded Z-gradient coil, we have used the following experimental parameters ... 

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