Loop-Storage

Figure 12 Schematic representation of 2D chromatography using an eight-port injection valve and two storage loops. (A) In the first position of the valve the storage loop 1 is loaded with the HPLC eluent, while the content of the storage loop 2 is analyzed by SEC. (B) In the second valve position storage loop 2 is loaded with HPLC eluent and the content of loop 1 is analyzed according the molecular size of the solute.

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],... 

When a conventional column is used as a first-dimensional column, two different LCxLC configurations may be used, with either two trapping columns or fast secondary columns in parallel rather than storage loops. In the former setup, each fraction from the first dimension is trapped alternatively on one of the two trapping columns. At the same time, the compounds retained from the previous fraction on the other trapping column are back-flushed onto the analytical column for second-dimension analysis. In the latter setup, a fraction from the first-dimension column is trapped alternatively at the head of one of the two columns during the loading step in a one-column... 

It is also possible to store fractions of the chromatogram intermediately in sample loops. From these storage loops the samples are transferred into the NMR detection cell, after when the separation has finished. The NMR measurement is then again carried out under static conditions. We refer to the combination of these two procedures as loop storage/loop transfer. 

In this case, the eluent is directly flowing from the chromatographic system into a storage device. As only selected peaks are measured in the NMR system the separation is monitored, in parallel, with an LC detector (typically a UV detector). A peak is selected from the chromatogram recorded by the LC detector, and the storage loop is isolated after a certain time delay which is necessary to allow the peak to move from the detector into the loop. Without interrupting the separation, further peaks can be trapped in the subsequent storage loops. 

Peaks in storage loop Peaks in the NMR detection cell with... 

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. 

Finally, use of the loop-storage/loop-transfer technique provides an escape from this dilemma. The chromatogram is acquired without any interruption, the mass spectrometer acquires MS and MS2 spectra online, and provides spectroscopic information which allows reliable peak selection for the storage procedure. The MS unit is used above all as a very selective and sensitive detector. After the chromatographic separation, it is available for other purposes and the collected samples are then independently analysed in the NMR spectrometer. 

The application of the mass spectrometer in the loop storage/loop transfer mode makes the best use of both systems. The data provided during the chromatographic stage can be used to identify the peaks and to ensure that the NMR spectrometer time is not wasted. The mass spectrometer is not blocked during longer-running NMR experiments. 

The void volumes in a coupled system are relatively high. The NMR probe, or the storage loop which is located further downstream, is reached ca. 1CM0 s after the peak first appears in the LC detector. The software must calculate such delays depending on the system parameters and the actual flow rate of the separation. When the peak has reached the desired position, the necessary actions for storage or measurement must be started. 

Even if the user is present to select the peaks manually, an automation routine is very useful. If the delay time for the peak to pass between the LC detector and the NMR probe or storage loop is short, it is difficult to define the peak maximum manually. 

Calculation of the timings for the movements of the peak between the different positions in the hyphenated system. The time taken for the peak to reach the NMR unit or the storage loop depends on the void volume between the LC detector and the NMR spectrometer loop. This is also a function of the flow rate. For precise and reproducible positioning of the peaks, the software must allow interactive selection of the peaks from the chromatogram and the automatic calculation of these time delays based on the actual parameters. 

For the loop-storage working mode, an additional instrument containing the storage loops needs to be under automatic control. 

The H LC-NMR spectra were obtained on peaks stored in the BPSU-36 storage loops. Data were acquired with WET [41] solvent suppression on the residual water and acetonitrile signals. A composite 90° observe pulse, (tt/2)y—(tt/2) x—(tt/2) y—(tt/2)x, was employed. Spectra were collected into 32K data points over a width of 12 019 Hz, giving an acquisition time of 1.36 s, with an additional relaxation delay of 1.5 s. The data were multiplied by a line-broadening function of 1 Hz to improve the signal-to-noise ratio and zero-filled by a factor of two before Fourier transformation. 

The next phase of the experimental procedure was to collect in storage loops the peaks of interest as determined by the continuous-flow 19F LC-NMR-MS experiment. Storage in the loops was triggered by the UV response and desired M+D ion. 

FIGURE 13.7 Instrumental setup of (a) a GCxGC system (Anon., GC x GC Comprehensive Two-Dimensional Gas Chromotography Form No. 209-184 R2.58-REV-1 LECO Corporation, St Joseph, MI 49085 P3, 2008. With permission.) (b) a LC x LC system based on a second-dimension column with two storage loops (T. Hyotylainen, LC x LC switching valves configuration Chromedia Amsterdam P Two Dimensional LC (LC x LC), 2008. With permission.). 

A fully automated two-dimensional (2D) chromatographic system was developed by Kilz et al. [91-93]. It consists of two chromatographs, one which separates by chemical composition or functionality and an SEC instrument for subsequent separation by size. Via a storage loop system, fractions from the first... 

The most sophisticated way of coupling the two chromatographs is a fraction transfer system comprising one eight-port injection valve (see Fig. 15). With two storage loops, it is possible to collect fractions continuously without losses. When starting the separation, loop 1 is in the LOAD position, whereas loop 2 is in the INJECT position. Each of the loops has a volume of 100-200 pi. When the first fraction leaves the HPLC system, it enters loop 1 and fills it. When loop 1 is completely filled with the fraction, the injection valves automatically switch to the opposite positions, i.e. loop 1 is then connected to the SEC system in the... 

Fig. 15. Schematic representation of 2D chromatography using an eight-port injection valve and two storage loops...

An important feature for such an automated system is the proper coordination of the flow rates of the HPLC and the SEC systems. Since fractions are continuously collected from HPLC and subjected to SEC, the collection time of one fraction must exactly equal the analysis time in the SEC mode. Depending on the number and size of the SEC columns, about 7-15 min are required for one SEC analysis. The flow rate in HPLC must be such that one storage loop is filled exactly within this time. In practice, typical flow rates are 20-40 pl/min and 1.5-2.5 ml/min for the first and second dimensions, respectively. 

Fig. 12.4 Instrumental set-up for the HPLC-NMR hyphenated approaches, (a) Peak sampling unit using storage loops, (b) Peak trapping onto SPE cartridges with parallel MS and C70genically cooled NMR detection [113], Source Reproduced with permission of Wiley.

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