Sample Preparation and Locking Spectrometer

Sample Position

  • the NMR tube containing sample is correctly positioned in the spinner by using the depth gauge before putting sample in magnet.
  • the gauge should always be set on 67 for standard samples
  • for Shigemi tubes and samples less than 0.6 ml, there is a 2nd depth gauge that can be adjusted to position your sample in the center of the field. It is stored in a drawer to the left of the 800 CPU.

Biological Samples (proteins & DNA)

  • 0.5 mM protein good for Nhsqc; 1.0 to 2.0 mM better for 3D
  • pH 5.0 best for protein NH groups, may loose some NHs with higher pH up to 6.5
  • low to moderate ionic strength buffer of your choice, keep EDTA at 0.1mM or less
  • 10% D2O (or at least 5% D2O) for lock
  • add 5 to 10 uL of 15 mM DSS for chemical shift referencing
  • 0.6 ml total volume in NMR tube is optimal (make 0.62 ml sample in Eppendorf tube)
  • can decrease volume to 0.52 ml if needed, but it may become difficult to shim
  • there is some sensitivity loss over 0.7 ml, so don’t use 1.0 ml samples
  • Shigemi tubes take 0.275 ml

Locking on 10% D2O

  • adjust Z0 to maximize lock signal, then activate lock
  • adjust lock phase to maximize lock signal
  • lock power should be at about 40; the lock will start to saturate above power of 40
  • lock gain should be about 40-45, which should then give 50-60% lock level
  • if lock does not respond to knobs, deactivate tuning circuit, recable to normal operation, then type ‘su’ at console and wait for beep. Then try tuning again.

Tuning Probe

  • attach proton probe cable to tuning circuitry
  • activate tuning circuitry by selecting channel 1 (this is 1H channel)
  • set tuning gain to 8; initially use lower setting if reading is off scale
  • tune 1H using knobs on bottom of probe to minimize reflected power
  • minimum should be below 10 (with gain on 8), but will be higher if high ionic strength
  • set tuning selector back to 0 (zero) before disconnecting probe cable