Cryoprobe safety and operating notes

Our 600 and 800 MHz cryoprobes have much in common, even though they are produced by different vendors. For safe operation:

• Read and be familiar with the details of the manufacturer's requirements and recommendations for working with the probes.
  • Varian
  • Bruker
• The allowed high power pulse widths and decoupling fields are set up on nonpolar test samples. The operating power is capped at this level and the pulse widths on real samples float, depending on the salt concentration in the sample. The Bruker "power check" feature will prevent the pulse sequence from executing if the high power limits are exceeded. The cryogenic power limits in BioPack will warn you if you are exceeding the limits, but do not stop the experiment.
• The amount of time that carbon and nitrogen decoupling is on must be limited to avoid putting too much heat into the decoupling coil. The vendors express those limits differently but the end result is almost the same. All standard parameter sets on both instruments come up with a pre-acquisition delay (d1) of 1 second and acquisition times (aq or at) between 70 and 90 milliseconds. Under these conditions, the decoupling is limited to a duty cycle of 8% or less. This ratio cannot be increased. You cannot make d1 shorter, and if you make aq/at longer, d1 must be correspondingly longer. The safe decoupling power also may drop as aq/at gets longer. The safe course of action is to stick with the standard parameters.
• For sequences that use simultaneous decoupling of carbon and nitrogen, the power on each channel must be reduced by half. (This limitation applies to all probes, warm or cold.) This corresponds to reducing the power level by 3 dB and increasing the decoupling pulse width by 40%. In the Varian software, this should be done for you automatically; in the Bruker software, it must be done by the operator. It is helpful to use adiabatic decoupling at the lower power in order to recover as much decoupling bandwidth as possible. Adiabatic decoupling on both channels is standard in the current BioPack software. Instructions for converting to adiabatic decoupling for carbon in the Bruker TopSpin software are available in the NMR Guide--from the top menu bar, choose Help, NMR Guide, and search "adiabatic decoupling." The pulse shape and decoupling sequence are precalculated and are available on the corresponding menu lists. Because the nitrogen chemical shift range is limited, decoupling bandwidth is less critical there. Our standard nitrogen garp decoupling pulse is 180 usec and increasing it to 250 usec with 3 dB less power is usually well tolerated.
• One additional point with the Varian cryoprobe: note the probe heater power before the experiment starts (typically about 5 watts). The pulse sequence should not drop this value by more than 1 watt in the first few minutes. If it does, stop the experiment and determine where the excess heat is coming from.
• One additional point with the Bruker cryoprobe: the sample depth is strictly limited. Probe damage will result if the bottom of the tube goes below the 21 mm mark. We set up the depth guage and our shims around a sample position of 19 mm.

For optimal spectra:

• You will get better water suppression in a Shigemi tube.
• With sequences that start on amide protons, you will get better water suppression from the sequences that use a flipback pulse. With sequences that involve alpha protons, flipback pulses are less desirable because they are not perfectly selective and some alpha signals will be very close to the water. If additional water suppression is needed, it may be better to use very low power presaturation.
• Because of radiation damping, the optimal values of flip-up and flip-down flipbacks may be quite different from each other and from the values extrapolated from the hard pulse calibration. The BioPack calibrations page has a flipback calibration button which will independently calibrate them. In the Bruker software, it may be helpful to set up the experiment, set the receiver gain, and type gs. Go to the "Shape Power" tab and test the effects of changing sp1. If you make a significant reduction in the water signal, save the new value of sp1, and re-run rga.
• Especially for long experiments, use lots of dummy or steady state scans (at least 5 minutes' worth) to let the probe and sample reach temperature equilibrium and establish optimal shims before taking the real data. You may want to engage z1 autoshimming and let that track the temperature changes during the dummy scans; otherwise, you may want to shim by hand under pulsing conditions. One challenge with this is the constant deflection of the lock because of the gradient pulses. The Varian software has a button called "Activate disabling gradient option." This allows you to turn the gradients off, start the experiment, and shim after the sample reaches steady state. Then stop the experiment, enable the gradients, and start your real data acquisition.

  Dave Vander Velde 11/23/05