Sample Preparation and Water Suppression

TMS cannot be used in aqueous solution because it is not water soluble. For a chemical-shift reference, a water-soluble equivalent (such as sodium d4-3-trimethylsilylpropanoate ((CH3)3SiCD2CD2C02-Na l, TSP )) can be added the single H peak is defined as zero ppm in water. The water peak itself can also be used as a chemical-shift reference, but care must be taken to correct for the temperature dependence of its chemical shift. Referencing of 13C and 15N chemical shifts can be done by using an accurate H reference. If the exact chemical shift is known at the center of the H spectral window (usually the water resonance), the precise radio frequency can be calculated for the zero point of the XH chemical-shift (ppm) scale. For example, on a 600 MHz spectrometer with a reference frequency of 600.13231564 MHz and a water chemical shift of 4.755 ppm  

It is important to use the most accurate values available for these ratios and to do the calculation on a computer spreadsheet to avoid truncation errors. Once you have the frequency corresponding to 0 ppm, the chemical shift at the center of the spectral window ( the carrier ) can be calculated from the reference frequency of the 13C or 15N channel as we did above for the ll channel. 

Radiation damping is strongest when the probe is matched perfectly and has a very high Q factor (a very sharp tuning dip ), so one way to minimize it is to detune the probe a bit. 

Another commonly used technique is the water flip-back pulse, a shaped pulse designed to selectively rotate only the water magnetization by 90°, putting it back on the +z axis after a hard (nonselective) pulse has rotated all of the sample magnetization into the x-y plane. Water can be viewed as a wild and powerful bucking bronco—it must be tamed and never allowed to get out of its pen. The best place for water is on the +z axis where it will not do any harm. This is the rationale behind the flip-back pulse every time water is moved from the +z axis, use a selective pulse to put it back there. 

1 Sequence-Specific Chemical Shifts in Structured Proteins 

---