Sample Preparation and Locking Spectrometer

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.

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

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.

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

Alabama High Field NMR Center