Spectroscopy Focus LC-NMR – Who in Their Right Mind Would Even Consider it?


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Bernie Monaghan (BM): Could you tell us a little about the group in which you work in Sunderland?


Nicolas Haroune (NH): Chemispec was established in 2000 when the University obtained a grant from the ERDF (European Research Development Fund) to purchase analytical equipment with the aim of helping small and medium enterprises in the North East of England. 2 NMR spectrometers (500 and 300MHz), 1 mass spectrometer (Apex II FT/MS) were purchased. More particularly, the 500MHz spectrometer was equipped with a flow probe for LC-NMR applications. We have now been offering LC-NMR analysis services nationwide for nearly 10 years to industries in the pharmaceutical and chemical sector. We have now been offering LC-NMR analysis services nationwide for nearly 10 years to industries in the pharmaceutical and chemical sector.


BM: Which manufacturers Instruments do you use currently? Are there any technological reasons for making these choices?


NH: These instruments were purchased from Bruker Ltd, a market leading manufacturer in this area.


LC-NMR is a powerful technique to obtain detailed structural information on organic compounds in complex mixtures and/or at low level. The data obtained are complementary to LC/MS data.


BM: Can you give the readers some background into the interest and need for such a technique? What are the practical operating issues that have to be overcome to obtain high quality data?


NH: Our LC-NMR equipment allows the detection of only microgrammes quantities of unknowns. It also allows:


• The detection of all protons containing analytes


• It is a non invasive technique so it is possible to recover the sample after analysis


• It provides detailed structural information which are complementary to mass spectrometry analyses


• It represents a time saving since there is no need to collect fraction and as such the sample preparation minimised


These advantages make the LC-NMR suitable for the analysis of components in complex mixture at low concentrations, where difficult to purify or to unstable for isolation by Silica gel chromatography or preparative HPLC.


A schematic view of the LC-NMR system is shown in Figure 1.


However, the enhancement of the quality data is spectacular as minimum background signals are observed; also these solvents are free from any impurities which can be found in organic solvents and again, can crowd the spectra with unwanted signals.


Finally, the peak of interest in the LC chromatogram should be well resolved and separated from major components, otherwise, severe cross contamination may occur in the NMR flow cell. To a certain extent, this can be overcome by storing a ‘slice’ of the LC peak in one of the loops of the BPSU (Bruker Peak Sampling Unit), and then eluting it towards the NMR at the end of the chromatography separation. However, this may not be suitable for unstable components, in which case the flow of the LC may be diverted to the waste using the switching valves of the BPSU, except when the compound of interest comes off the column.


BM: What advantages does NMR hold in identifying the active ingredients? Do you have examples where other detection methods alone were insufficient for your needs?


NH: NMR allows the scientist to obtain very detailed structural informations on the environment of the different protons in a given molecule, which are not easily obtainable by other techniques such as mass spectrometry (MS). For example, oxidation of an active ingredient is routinely identified by MS during stability trials of formulations. However, determining the exact position of this hydroxylation in the molecule is not always straightforward by MS/MS, especially when this takes place on aromatic rings. A number of these issues have been tackled by the use of LC-NMR.


BM: Are there other detection methods that you would like to include in your instrumentation? ELSD for example?


NH: Evaporative light-scattering detection (ELSD) is regarded as a valuable alternative to UV detection for liquid chromatographic analysis of substances that do not contain a chromophore. A key feature of ELSD is that - unlike refractometry - it can operate in gradient mode, thus allowing application of more selective liquid chromatographic methods. However, LC-NMR analysis requires the sample to be liquid when reaching the flow probe so, unless the flow is split prior the NMR probe, the destructive nature of light scattering detection prior NMR detection would be inappropriate.


BM: Do you have examples separations run by the system that the readers would find impressive.


NH: We have several excellent examples but unfortunately they are subject to confidentiality agreements but we hope to print and share these results some time soon.


Figure 1. Schematic of Instrument layout. Author Details:


Dr Nicolas Haroune, ChemiSPEC, University of Sunderland UK nicolas.haroune@sunderland.ac.uk


LC-NMR separation is usually carried out with a mixture of deuterated water and protonated organic solvent. It is fine to use these when the 5 to 10ppm region is of interest but since these solvents give rise to strong signals in the 1 to 5ppm region, they can potentially overlap signals of interest.


This problem can be overcome by using fully deuterated organic solvents but these are expensive ~£650/500mL.


Meantime Dr Karine Ndjoko Ioset from the Laboratoire of Pharmacognoxy and Phytochemistry from the University of Geneva, Switzerland has published work and presented an overview of the technique of LC-NMR at the spring symposium of The Chromatographic Society [1] held in Sunderland, UK in May 2009. Here she concentrated on extracts from natural products and the use of hybrid NMR techniques to identify previously unidentified (and often the most physiologically active) components of the extract.


The advantages of using are that the full structural and stereochemical information can be obtained (by the use of 2D NMR) but also it is a highly non-selective detection technique. Several examples were shown where assisted in the identification of unknown compounds such as those shown in Figure 2.


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