Stop-flow mode

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

FIGURE 7.8 Schematic of HPLC-NMR setup illustrating the pathways used for on-flow and stopped flow modes (pathway A) and loop collection mode (pathway B), shown by the dashed line. 

LC-NMR is a powerful technique especially for the analysis of compounds which cannot be isolated by preparative HPLC. For the identification of impurities in API from unpublished work from this laboratory, impurities at a 1% level from drug substances can be identified on a 600 MHz NMR spectrometer using the stop-flow mode. 

Both the capillary LC and NMR are controlled by the interface software, which enables the operator to use the UV-detector output for peak selection. Only peaks of interest can be subjected to NMR analysis, while minor or unimportant compounds can be directed to waste. NMR acquisition can take place in either on-flow or stop-flow mode. The combination of capillary LC and NMR is suitable for sample-limited applications (e.g., proteomics) and allows for low nanograms detection. 

Carry-over can be problematic for closely eluting peaks in stop-flow mode [46], and these are better analysed using the loop collection mode (see below). 

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... 

Comprehensive 2D HPLC can be also operated under stop-flow mode. In this case, after transferring a desired fraction volume onto the secondary column, the flow of the mobile phase in the first dimension is stopped and the fraction analyzed in the second dimension. When the separation is finished, the mobile-phase flow in the first dimension is switched on and the whole procedure is repeated again for the analysis of all the transferred fractions. The disadvantage of this procedure is the long analysis time, while the advantage can be that the second-dimension column can give higher plate numbers if compared to the continuous approach [23]. 

LC-NMR can be operated in two different modes on-flow and stopped-flow. In the onflow mode, LC-NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60 p.1 cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20 pig. Thus, on-flow LC-NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible. 

The present performance of high field NMR instruments allows the recording of a spectrum in quantities of micrograms. Under these conditions, it is possible to install a flow cell of only a few microlitres into the magnet of the instrument which allows the spectra of the analytes to be recorded. The experiment is conducted with a very small flow rate of the mobile phase (D20 or CD3CN) or in the stop-flow mode. In this mode, the mobile phase is momentarily stopped in order to record the spectrum. This technique, which requires very expensive materials, is of limited use. It is used mainly to isolate and identify very unstable compounds that cannot be isolated through classical means. 

Define a 3 mm x 3 mm square working area using either PVC tape (batch mode) or Tippex correction fluid (stop-flow mode). 

Typical calibration plots obtained for progesterone in milk are shown in for batch mode (chronoamperometry) and for stop-flow mode (amperometry) in Fig. 35.4. 

Fig. 35.4. Calibration plots for progesterone in milk obtained using (A) batch mode or (B) stop-flow mode operation. (Reproduced with permission of Elsevier B.V. from Refs. [1] and [2], respectively.)...

The dynamic range in batch mode lies between 5 and 50ng/mL (Fig. 35.4A), whereas in stop-flow mode a lower range can be obtained of between 2 and 25ng/mL (Fig. 35.4B). 

System Operation LC-ARC operates in both the stop-flow and non-stop-flow modes. Each of these is described below. 

Stop-Flow Mode The LC-ARC StopFlow controller performs two major functions. First, the controller maintains the back pressure during stop-flow data acquisition. This function is crucial for maintaining the shape and resolution of a peak. The second function is to deliver a liquid scintillation cocktail necessary for peak detection by the radiochemical detector. 

There are two ways to operate the stop-flow mode. The by-level mode, which is based upon the defined count zone(s), operates by signaling the instalment to continue running until a radioactive peak above a designated level is detected. The instrument will then stop and count the predefined fraction (in seconds), which is defined in the count zone. A second way to operate the stop-flow mode is the by-fraction mode. The instalment will stop and count every fraction (in seconds) within the predefined count zone, regardless of whether any radioactivity is detected or not. The fraction size in the stop-flow mode and the volume of liquid scintillant used for counting can be automatically calculated by the instalment or the parameters can be defined by the user. 

Non-Stop-Flow Mode In the non-stop-flow mode, the LC-ARC system is operated in a similar manner to the conventional continuous-flow analysis. If a mass spectrometer is coupled to the LC-ARC system, the LC effluent is split postcolumn to deliver a fraction to the radiochemical detector and the balance to a mass spectrometer. 

The LC-ARC system, with the stop-flow and the non-stop-flow modes, provides a convenient platform for analysis of samples with a wide range of radioactivity. A major advantage of this technique, compared to other conventional in-line... 

The analytical NMR flow-cell (see Figure 1.8) was originally developed for continuous-flow NMR acquisition, but the need for full structural assignment of unknown compounds led to major applications in the stopped-flow mode. Here, the benefits of the closed-loop separation-identification circuit, together with the possibilities to use all types of present available 2D and 3D NMR techniques in a fully automated way, has convinced a lot of application chemists [17-70], A detailed description of the different modes for stopped-flow acquisition (e.g. time-slice mode) is found in Chapters 2 und 3. 

All three suppression techniques can be used either for stopped-flow or continuous-flow acquisition. Presaturation works quite well in the stopped-flow mode, whereas the WET sequence seems to be superior in the continuous-flow mode. 

Figure 2.3 Peak broadening effects of the (a) direct stop-flow and (b) loop-storage/loop transfer procedures. The amounts of washing solvent required is defined by the chromatographic separation of the peaks in the direct stop-flow mode, while being user-defined in the loop transfer mode...

The chromatographic stage is not interrupted and therefore no stop-start effects will create disturbances. The peaks are separated in the storage loops, and therefore the NMR measurement time is not limited and will not decrease the performance of other peaks. In complex chromatograms the chance of finding the peak(s) of interest is dramatically increased. As in the direct stop-flow mode, the static conditions provide stability and the best NMR conditions for the acquisition of all kinds of high-resolution ID and 2D NMR spectra. 

In the stop-flow mode, the additional time could be used to acquire MS spectra of higher order, although these experiments would still be finished long... 

This study evaluated the impurity profile of untreated water from a textile plant in Portugal [35]. The organic material was concentrated by extraction from 11 of water into dichloromethane and HPLC-NMR and HPLC-MS experiments were carried out using a reverse-phase separation with an acetonitrile/ D2O gradient elution with H NMR spectroscopic observation at 600 MHz. For the HPLC-NMR studies, the samples were further fractionated into two pools according to their HPLC retention times. The HPLC-NMR studies were carried out in the stop-flow mode and the combination of NMR and MS results yielded the identification or tentative identification of 14 compounds, comprising mainly surfactants, anthraquinone dyes and nonylphenol-related molecules. 

---