Time-slice detection

FIGURE 2.11 Comparison of time-slice detection and time-array detection. (Reprinted with permission from reference 10). 

There are four general modes of operation for LC-NMR on-flow, direct stop-flow, time-sliced and loop collection/transfer. The mode selected will depend on the level and complexity of the analyte and also on the NMR information required. All modes of LC-NMR can be run under full automation for LC peak-picking, LC peak transfer to storage loops or NMR flow cell, and NMR detection [46],... 

Time-shcing is a variation of the stop-flow mode where the flow is stopped in a time-dependent manner (e.g. every 15 s) for the whole chromatographic run, in a sense the ultimate approach in slow-flow. When the flow is stopped, sufficient scans can be made to give the desired level of detection before moving on to the next time-slice. The whole process can be automated through software control. There is no requirement for an UV detector, and the approach has been used to identify non-UV-active components in drug substances [48]. The peak purity of... 

Fig. 5. Strategies for isotope dilution analysis utilizing on-line coupling IC-ICP-MS. Shown is the separation of bromate and bromide by anion IC.The sample was 585 )Lll of a bottled water, spiked with bromate and a bromate isotope standard. The column, eluent and detection device are as described in Fig. 4. Shown are the mass traces form/z79 and 81 (dotted line) and the total time slice isotope ratio for m/z 79/81.

It is also worth briefly reiterating here the main operational modes for HPLC-NMR and HPLC-NMR-MS. There are currently five main options which can be employed for HPLC-NMR using either isocratic or gradient elution. These are continuous-flow, stop-flow, time-sliced stop-flow, peak collection into capillary loops for post-chromatographic analysis, and automatic peak detection with UV-detected triggered NMR acquisition. 

At time point t (in the rth time slice, meaning t° l < f < t(n), the density matrix can be calculated from // l according to Equation (43), and so the detected signal at time point t is given as ... 

Figure 11 Simulation of the fid of a spin set. (A) Individual density matrix is calculated at each exchange point. (B) Eigencoherence representation of the density matrix is propagated from the beginning of the time slice for each detection point.

The opportunity to measure the dilute polymer solution viscosity in GPC came with the continuous capillary-type viscometers (single capillary or differential multicapillary detectors) coupled to the traditional chromatographic system before or after a concentration detector in series (see the entry Viscometric Detection in GPC-SEC). Because liquid continuously flows through the capillary tube, the detected pressure drop across the capillary provides the measure for the fluid viscosity according to the Poiseuille s equation for laminar flow of incompressible liquids [1], Most commercial on-line viscometers provide either relative or specific viscosities measured continuously across the entire polymer peak. These measurements produce a viscometry elution profile (chromatogram). Combined with a concentration-detector chromatogram (the concentration versus retention volume elution curve), this profile allows one to calculate the instantaneous intrinsic viscosity [17] of a polymer solution at each data point i (time slice) of a polymer distribution. Thus, if the differential refractometer is used as a concentration detector, then for each sample slice i. 

Coupling of LC to NMR is relatively simple. The effluent from the column is delivered through a polyether-ether ketone (PEEK) transfer line to the NMR flow cell, which typically has a volume of 60 jA. The measurement can be carried out in one of four modes on-flow, stop-flow, time-sliced and loop collection. In the on-flow mode, the effluent from the column flows continuously through the NMR flow cell. Because of the very short time available for the measurement when peaks elute in real time, this approach is limited to major components of a mixture. In the stop flow mode, peaks detected with a UV detector are transferred to the NMR flow cell, and the run is automatically stopped. The NMR spectra can then be acquired over a period of several minutes, hours or even days. In the time-sliced mode, the elution is stopped several times during the elution of the peak of interest. This mode is usually used when two analytes are poorly resolved. In the loop collection mode, the chromatographic peaks are stored in loops for offline NMR study. This approach is therefore not a real online hyphenated technique. 

The detection limits of the on-flow mode can be improved by performing the analysis at low-flow (<0.1 ml min or by running time-slice experiments over a whole chromatogram. In this latter case, the flow is stopped at defined time intervals. Both modes of operation enable higher number of transients per increments to be recorded and thus a significant improvement in S/N ratio is obtained. 

Rgure 6 Comparison of the on-flow and time-slice LC/NMR contour plots of the MeOH extract of the aerial parts of Gnidia involucrata (Thymelaceae). As shown, the on-flow experiment allows the detection of four major compounds while the time-slice procedure reveals 20 components. Amount injected 20mg. HPLC conditions 018 column with radial compression, Waters RCM 8 X 10 (100 X 8 mm i.d.) MeCN-DaO gradient (5 95 to 20 80 in 50 min) 0.9 ml min k LC/NMR conditions 24 scans/increment (onflow) 1024 scans/increment (time-slice) 60 pi flow cell (3 mm i.d.) 500 MHz. (Reproduced with permission from Wolfender J-L, Ndjoko K, and Hostettmann K, (2001) The potential of LC-NMR in phytochemical analysis. Phytochemical Analysis 12 2-22 John Wiley Sons Ltd.)... 

Time-Slice Vs.Time-Array Detection. Holland et al. have referred to this method of data collection as time-slice recordinj. It is shown schematically in... 

Pali), P and Stamford, JA (1994) Real-time monitoring of endogenous noradrenaline release in rat brain slices using fast cyclic voltammetry. 3. Selective detection of noradrenaline efflux in the locus coeruleus. Brain Res. 634 275-282. 

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