Calibration of Offset Frequencies

The offset frequency (v0ffset) has the following relationship with the carrier frequency vcarr •

in which v^t is the base frequency of the instrument for the nucleus, which is the fixed frequency for a specific nucleus. For instance, in a 600 MHz instrument, uinst for 1H, 13C, and 15N is 599.5200497 MHz, 150.7747596 MHz, and 60.7557335 MHz, respectively.

4.3.4.1. Calibration of Transmitter Offset Frequency

Because common biological samples are in aqueous solution, the transmitter voffset (e.g., o1 on a Bruker instrument and tof on a Varian instrument) is required to be set on the water resonance for experiments with water suppression. If the transmitter voffset is off by even 1 Hz, it will significantly affect the result of water suppression experiments. Therefore, vtof calibration is performed for each individual sample. It is first estimated from the water peak using a one-pulse sequence by setting it on the center of the water peak. It is then arrayed by 0.5 Hz in the range of ±3 Hz of the setting value using a PRESAT sequence (see solvent suppression) with 2 transients for steady state and 8 transients per FID. The correct vtof gives the lowest intensity of the water peak (Figure 4.4a). If the 2H signal of 2H2O is adjusted to be on-resonance prior to 2H locking, vtof should be within the range of a few hertz for different samples at a given temperature. It may have very different values if the lock is not adjusted to be on-resonance prior to the calibration.

4.3.4.2. Calibration of Decoupler Offset Frequency

The correct decoupler offset frequency (e.g., o2, o3 on a Bruker instrument or dof, dof2 on a Varian instrument) needs to be known before pulse calibration for the decoupler channel. A simple one-pulse experiment with continuous wave (CW) heteronuclear decoupling is used for this purpose. The power of the CW decoupling is set very low so that only a narrow frequency range (a few tens of hertz) is decoupled in order to accurately determine the value of Vdof. Arraying the decoupler voffset in the experiment provides a series of spectra with modulated signal intensity (Figure 4.5). As the voffset becomes closer to the resonance frequency of the heteronuclear signal, the doublets become closer, resulting in an intensity increase of the peak. The maximum intensity is obtained when the voffset is on or near resonance. The lower the CW power, the narrower the frequency bandwidth it decouples. The spectra shown in Figure 4.5 were obtained with a CW RF field strength of 70 Hz. The maximum pulse power of a 300 watt heteronuclear amplifier was attenuated by -42 dB, which cuts down the pulse power by a factor of 128 (or 27). (As a reminder, attenuation by -6 dB decreases RF field strength by a factor of 2.) If the bandwidth of the CW decoupling is wider than the increment of the decoupler voffset array, there will be several spectra with the same maximum intensity as the peak. The average value of the voffset in the spectra with the same maximum intensity should be taken to get the calibrated decoupler offset frequency.

Figure 4.5. Arrayed spectra for calibrating decoupler offset frequency using 15N urea in DMSO. The decoupler offset frequency is arrayed with increments of 1 Hz as stated in the text. The spectrum labeled with an * has the offset frequency on urea 15N resonance.

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