Detection period

Eisenberg, D., Weiss, R.M., Terwilliger, T.C. The hydrophobic moment detects periodicity in protein hydropho-bicity. Proc. Natl. Acad. Sci. USA 82 140-144, 1984. 

All these experiments involve at least three distinct time periods preparation (tp), evolution (tl), and detection (t2) these periods are usually separate by rf pulses. Some experiments (e.g., NOESY, RELAY) further contain an additional "Mixing period, tm, between the evolution and detection periods. 

Figure 3.1 The various time periods in a two-dimensional NMR experiment. Nuclei are allowed to approach a state of thermal equilibrium during the preparation period before the first pulse is applied. This pulse disturbs the equilibrium ptolariza-tion state established during the preparation period, and during the subsequent evolution period the nuclei may be subjected to the influence of other, neighboring spins. If the amplitudes of the nuclei are modulated by the chemical shifts of the nuclei to which they are coupled, 2D-shift-correlated spectra are obtained. On the other hand, if their amplitudes are modulated by the coupling frequencies, then 2D /-resolved spectra result. The evolution period may be followed by a mixing period A, as in Nuclear Overhauser Enhancement Spectroscopy (NOESY) or 2D exchange spectra. The mixing period is followed by the second evolution (detection) period) ij.

There are actually two independent time periods involved, t and t. The time period ti after the application of the first pulse is incremented systematically, and separate FIDs are obtained at each value of t. The second time period, represents the detection period and it is kept constant. The first set of Fourier transformations (of rows) yields frequency-domain spectra, as in the ID experiment. When these frequency-domain spectra are stacked together (data transposition), a new data matrix, or pseudo-FID, is obtained, S(absorption-mode signals are modulated in amplitude as a function of t. It is therefore necessary to carry out second Fourier transformation to convert this pseudo FID to frequency domain spectra. The second set of Fourier transformations (across columns) on S (/j, F. produces a two-dimensional spectrum S F, F ). This represents a general procedure for obtaining 2D spectra. 

In three-dimensional experiments, two different 2D experiments are combined, so three frequency coordinates are involved. In general, the 3D experiment may be made up of the preparation, evolution (mixing periods of the first 2D experiment, combined with the evolution t ), mixing, and detection ( ) periods of the second 2D experiment. The 3D signals are therefore recorded as a function of two variable evolution times, t and <2, and the detection time %. This is illustrated in Fig. 6.1. 

SELINQUATE (Berger, 1988) is the selective ID counterpart of the 2D INADEQUATE experiment (Bax et al., 1980). The pulse sequence is shown in Fig. 7.4. Double-quantum coherences (DQC) are first excited in the usual manner, and then a selective pulse is applied to only one nucleus. This converts the DQC related to this nucleus into antiphase magnetization, which is refocused during the detection period. The experiment has not been used widely because of its low sensitivity, but it can be employed to solve a specific problem from the connectivity information. 

Detection period The FID is acquired in the last segment of the pulse sequence. This is the detection period, and, in 2D experiments, this is the <2 domain. 

The separation of interactions by 2D spectroscopy can be compared with 2D chromatography. In a onedimensional thin layer or paper chromatogram, the separation of the constituents by elution with a given solvent is often incomplete. Elution with a second solvent in a perpendicular direction may then achieve full separation. In NMR spectroscopy, the choice of two solvents is replaced by the choice of two suitable (effective) Hamiltonians for the evolution and detection periods which allow unique characterisation of each line. 

It must be stressed that the spin-echo sequence is applied only during the detection period and its unique purpose is to estimate the signal amplitude (in a sense, it is a replacement for the simple 90° pulse). Consequently, in an arrayed multi-block experiment whose purpose is to measure Ti(Br), only the X value is varied, while the delays 5 and 8 are kept constant in order to make sure that no T2(Ba) effects leak into the experimental relaxation curves. Moreover, to avoid contamination of the echo by FID residues due to imprecise settings of RF pulses and to Bi inhomogeneity, proper phase cycling is highly recommended. 

Fig. 27. Thermally balanced PP and NP sequences. (PP) In the balanced PP sequence, the sample is first kept at the relaxation field By for a time — t and, then pre-polarized at the polarization field Bp for a time Tp, and finally allowed to relax for time T before the start of the detection period. The time Tp should be set to about 4Ti(Bp). As T varies during a multi-block sequence, the polarization interval position moves horizontally but the total block duration and the mean power dissipation remain constant. (NP) The balanced non-polarized sequence is conceptually similar, except for the fact that the polarization interval is replaced by a magnetization annihilation interval in which the field is zero and whose duration should be about 47 (0). In both cases, the time should be about or more than 4Ti(Br). The concept can be combined with any detection mode, not just the simple FID detection shown here.

A number of 2D NMR experiments have been developed which allow the study of slow exchange phenomena. The most common (Exchange Spectroscopy, EXSY), is based on the standard pulse sequence 90°x-fi-90°x-tm-90°x - FID(t2), where is an evolution delay, is the mixing time, and tj is the detection period [175, 176]. 

Fig. 1. Pulse sequence of the C HSQC experiment with a spin-lock pulse for the suppression of signals from protons not bound to C. Narrow and wide bars denote 90° and 180° pulses, respectively. The spin-lock pulse is labeled SL. r is set to 1/[2J( C, H)]. The detection period is symbolized by a triangle. Phase cycle ] = 8(y) 4>2 = 2 x,x,y,y) 03 = 4 = 4n = 8(x) 05 =4(x,—x) 05 = 4(x),4(—x) acquisition = 2(x,—x,—x,x). The phases of the C pulses before U (03 and 0.5) are subjected to the States-TPPI scheme [38].

An experiment intended to measure a relaxation rate consists in general of three elements the preparation period, the relaxation period and the detection period. The scheme differs a little from the famous four-period division of two-dimensional experiments [7]. In the case of two-dimensional... 

The preparation period consists of the creation of a non-equilibrium state and, possibly, of the frequency labeling in 2D experiments. Usually, the preparation period should be designed in such a way that in the created non-equilibrium state, the population differences or coherences under consideration deviate as much as possible from the equilibrium values. During the relaxation period, the coherences or populations evolve towards an equilibrium (or a steady-state) condition. The behavior of the spin system during this period can be manipulated in order to isolate one specific type of process. The detection period can contain also the mixing period of the 2D experiments. The purpose of the detection period is to create a signal which truthfully reflects the state of the spin system at the end of the relaxation period. As always in NMR, sensitivity is a matter of prime concern. 

As previously mentioned, the detection period for a drug depends on a number of factors, including the type of opiate, the type of sample, the frequency of drug use, metabolic rate, age, body mass, drug tolerance, and overall health. Generally speaking opium can be detected for 5-7 days after its use. Other opiates such as heroin and codeine have significantly shorter detection periods (Table 10.3). 

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