LC-NMR Automation

There has been significant advancement in the applications of NMR to the development of small-molecule pharmaceutical products. For example, advances in NMR automation (e.g., flow-injection analysis) and directly coupled methods (e.g., LC-MS-NMR analysis) have made analysis and characterization of small-molecule drugs much easier.23 25 These improvements have helped chemists to develop and characterize small-molecule combinatorial libraries and to screen for active compounds.4 6 It is likely some of these techniques can also be used in biopharmaceutical product development. 

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],... 

Automation of LC-NMR is now at a stage where the operator can inject a sample and leave the HPLC interface to detect and store peaks and the NMR spectrometer to collect one- and two-dimensional data with signal-to-noise-dependent collection. An example of automated loop collection and transfer of closely eluting peaks is shown in Figure 6.38. Structures were deduced from the aromatic peak patterns and LC-MS information. Peaks 1-5 all elute within 5 min with no carry-over present in any of the H spectra. 

The number of impurities requiring identification. The automation routines offered by LC-NMR gives large efficiency gains when there are several impurities present in a sample (or multiple samples). 

Equipment available (HPLC, automated PSU, SPE, flow probe, high-field spectrometer) will determine the overall LC-NMR sensitivity and levels of impurities that can be analysed. For low-level impurities the time required for multiple trapping on SPE cartridges may also need to be considered [84]. 

The coupling of LC (liquid chromatography) with NMR (nuclear magnetic resonance) spectroscopy can be considered now to be a standard analytical technique. Today, even more complex systems, which also include mass spectrometry (MS), are used. The question arises as to how such systems are handled efficiently with an increasing cost and a decreasing availability of skilled personal. LC-NMR and LC-NMR/MS combine the well-established techniques of LC, NMR and MS. For each of those techniques, various automation procedures and software packages are available and used in analytical laboratories. However, due to the necessary interfacing of such techniques, completely new demands occur and additional problems have to overcome. 

Following our discussions of the principle differences between the various working modes of LC-NMR, we will now describe the practical aspects of the measurements, and in particular the possibilities of using automation routines. 

In LC-NMR this problem does not arise in the same way as it does in standard sample changer automation systems, where a change between such different solvents as water and chloroform is possible. In LC-NMR, the samples are eluted from a column in a mixture of solvents, in which only the relative composition changes ... 

Changing solvents also has an effect on the homogeneity of the magnetic field. Again, the observed changes in LC-NMR systems are not as large as those found in normal sample changer automated devices ... 

Automation of the above LC/NMR procedures has now advanced to a stage where, with the appropriate interface and software, the analyst can inject a sample and walk away, leaving the LC interface to detect and store (or not) peaks appropriately, and the NMR spectrometer to collect data with S/N-dependent collection of 2D data as desired. 

Due to the lack of sensitivity, most of the LC/NMR applications requires the accumulation of many transients for achieving a satisfactory detection of the compounds of interest. For the recording of detailed structural information, measurement of 2D correlation experiments is also often mandatory. Consequently, LC/NMR can be operated not only in dynamic but also in static conditions. These main operation modes are described below and require different level of automation ... 

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