Decoupling Hardware

How does a spectrometer deliver this RF irradiation to the probe? Compared to normal excitation pulses, which are very high-power and short (~10 |xs) duration, decoupling requires low-power irradiation for the entire acquisition time (1-2 s). This is usually accomplished by having two separate sources of RF power, a "broadband" transmitter that can be

Figure 4.7

operated at a wide range of frequencies (e.g., 15N is 30.4 MHz, 13C is 75.4 MHz, and 1H is 300.0 MHz on a 7.05 T instrument) and a proton decoupler that can only produce the proton frequency (e.g., 300.0 MHz). The transmitter is set to the frequency of the nucleus to be observed with a high-power level for pulses, and the decoupler is set to a low-power level for proton decoupling. As soon as the pulse sequence is over and acquisition of the FID begins, the decoupler is turned on for the duration of the acquisition time. Even when 1H is being observed, the proton (high-power) pulses come from the transmitter, and the decoupler is used to deliver the low-power 1H irradiation during the acquisition time. This is necessary in older machines because it takes time (milliseconds!) and requires physical switching of relays to change the power level of the transmitter, so you can not just use a single source of RF to supply high-power pulses and low-power decoupling irradiation.

Waltz-16 Figure 4.9

With instruments that are a bit more modern than this basic system, the decoupler is not fixed at the frequency. Instead, it is identical to the transmitter in that it can be set to the frequency of any nucleus. Thus, you could decouple 13C while observing !H, for example (an "inverse" experiment). This arrangement is called "dual broadband" because both RF sources are "broadband"—adjustable over a wide range of frequencies. Still more modern spectrometers can switch power levels in a few microseconds without relays, so that a single "box" can be used for all proton RF, whether it is for high-power pulses or for low-power decoupling. This feature makes the whole concept of "transmitter" and "decoupler" a matter of language rather than real hardware differences. Figures 4.8-4.10 show the configuration of a Varian Unity-300 spectrometer (with direct 13C probe) for routine 1H, for 13C with 1H decoupling, and for 1H with homonuclear decoupling. Note that the inner coil of the probe is used for 13C to maximize the sensitivity of detection of this "insensitive" nucleus. The outer coil, which is farther from the sample and therefore less sensitive, is used for 1H because it is much easier to detect.

For routine 1H acquisition (Fig. 4.8), the transmitter is set to the 1H frequency (300 MHz) and pulses from the transmitter are directed to the outer coil of the probe, which is tuned to the 1H frequency. The decoupler is not used. After the exciting pulse, the 1H FID is detected on the outer coil of the probe and directed to the receiver, which uses a continuous signal from

the transmitter (300 MHz) as a reference frequency that is "mixed" with (i.e., subtracted from) the FID frequency. For routine 13C acquisition with 1H decoupling (Fig. 4.9), the transmitter is set to the 13 C frequency (75 MHz) and pulses from the transmitter are directed to the inner coil of the probe, which is tuned to the 13 C frequency. The decoupler operates continuously at low power (with waltz-16 phase modulation) and its output is directed to the outer coil of the probe, which is tuned to the 1H frequency. After the exciting pulse, the 13C FID is detected on the inner coil of the probe and directed to the receiver, which uses a continuous signal from the transmitter (75 MHz) as a reference frequency that is "mixed" with (i.e., subtracted from) the FID frequency. For 1H acquisition with selective 1H decoupling or NOE difference (Fig. 4.10), the transmitter is set to the 1H frequency (300 MHz) and pulses from the transmitter are directed to the outer coil of the probe, which is tuned to the 1H frequency. The decoupler operates continuously at very low power during the relaxation delay (NOE difference) or during the acquisition of the FID (selective homonuclear decoupling) and its output is also directed to the outer (1H) coil of the probe. In either case, the 1H FID is detected on the outer coil of the probe and directed to the receiver, which uses a continuous signal from the transmitter (300 MHz) as a reference frequency that is "mixed" with (i.e., subtracted from) the FID frequency.

Changing from one of these configurations to another simply involves changing the frequency settings of the two channels ("transmitter" and "decoupler") and rerouting the outputs to the probe inputs. This is done by resetting electrical relays, which give a "click" when you issue the command (Varian: su or go, Bruker: ii or zg) to set the hardware according to the experimental parameters. Figure 4.11 shows the configuration for an "inverse-mode" experiment (Chapter 11), in which 1H is detected and pulses are delivered to the probe on both the 1H and 13C channels (e.g., 2D HSQC, a 1H-detected 2D 13C-1H correlation experiment). In this case an inverse probe is used, which has the inner coil tuned to 1H (the "observe" nucleus) and the outer coil tuned to 13C (the "decoupler" nucleus). The transmitter is set to the 1H frequency (300 MHz) and pulses from the transmitter are directed to the inner coil of the probe, which is tuned to the 1H frequency. The decoupler is set to the 13 C frequency (75 MHz) and pulses from the decoupler are directed to the outer coil of the probe, which is tuned to the 13C frequency. After the HSQC pulse sequence, the 1H FID is detected on the inner coil of the probe and directed to the receiver, which uses a continuous signal from the transmitter (300 MHz) as a reference frequency that is "mixed" with (i.e., subtracted from) the FID frequency.

Pulse

Modern NMR spectrometers may have many sources of RF ("channels"). For biological NMR, it is typical to have three channels, usually set to the frequencies of XH (e.g., 600 MHz), 13C (e.g., 150 MHz), and 15N (e.g., 60 MHz). Bruker refers to these as F1, F2, and F3, whereas Varian uses transmitter, decoupler A, and decoupler B. Recently, a fourth channel has become common, used for decoupling of deuterium (2H) in 2H-labeled proteins and nucleic acids. In these 3-channel and 4-channel spectrometers, the RF for pulses and for the reference frequency is produced by separate fully broadband (zero to 1H frequency) sources, and these low-power (~1 V) signals are fed into power amplifiers to boost them to the high power (50-300 W) needed for pulses fed into the probe. The power amplifiers are not broadband: one is devoted to 1H and the others are for all nuclei except 1H ("X" nuclei). For example, on a 3-channel 600-MHz spectrometer, one power amplifier handles only 600 MHz (1H) pulses, whereas the other two are broadband from 6 to 242 MHz (242 MHz is the 31P frequency, the highest frequency below 1H (except 19F, 570 MHz)). The interface between the flexible low-power RF sources and the more restricted power amplifiers is a kind of switchboard, whose connections depend on which nucleus is being detected and what kind of experiment is being done. The connections in the switchboard are not made by moving cables or switching physical relays, but rather by solid-state switches (PIN diodes) that are controlled by software. These switches can be changed in 1 ^s or less and do not have moving parts to wear out like relays.

4.6 DECOUPLING SOFTWARE: PARAMETERS

Most Varian decoupling parameters start with the letter "d" to distinguish them from the transmitter parameters, which start with a "t." Bruker uses a "1" (F1 channel) to specify the transmitter channel and a "2" (F2 channel) to specify the decoupler channel. The following parameters can be examined by entering dg (Varian) or eda (Bruker):

Bruker

Varian

nuc2

dn

o2

dof

dm

pl17

dpwr

cpdprg2

dmm

dcpd2

dmf

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