Hyphenated techniques

Abstract

To fulfil the need for efficient and rapid on-line identification of peaks from a chromatographic separation the hyphenation of chromatographic separation techniques with spectroscopic identification techniques is of increasing importance.The separation technique usually applied is high-performance liquid chromatography; the most useful spectroscopic techniques for elucidation of the structures of unknown compounds are mass spectrometry and NMR spectroscopy. Hyphenation of these two spectroscopic techniques with HPLC will, therefore, furnish extremely valuable information.Within the field of mass spectrometry new powerful ionisation techniques have been developed. Electrospray ionisation (ESI) and atmospheric-pressure chemical ionisation (APCI) can be easily coupled with HPLC and proved to yield extremely valuable information [1].Within the field of NMR spectroscopy the application of continuous-flow probes with a detection volume of either 60 or 120 µL is now routine, with over 200 installations world-wide. The on-line coupling of HPLC and NMR can be performed in either the stopped-flow or continuous-flow mode. Current sensitivity levels are in the 500 ng range for one-dimensional 1 H NMR spectra and in the microgram range for two-dimensional spectra.Combination of effective on-line coupling of HPLC with ESI MS, APCI MS, and NMR has been shown to be extremely helpful for the solution of a myriad of separation and identification problems in pharmacy, biotechnology, and nutrition [2, 3, 4].Miniaturisation of on-line coupling techniques to meet the needs of effective research in the new millennium is a major challenge. Whereas the use of capillaries is becoming more popular in HPLC-MS, capillary HPLC-NMR is still under development [5,6,7,8,9]. Figure 1 shows a sensitivity test of a 2.0-mm capillary HPLC-NMR probe with a detection volume of 750 nL. This newly designed probe with a double-saddle Helmholtz coil results in significant improvements in signal line shape and easy magnetic-field homogenisation. The signal-to-noise ratio of 50:1 obtained for the anomeric proton of 0.2 mol L -1 solution of sucrose in D 2 O is sufficient for elucidation of the structures of naturally occurring substances.Besides HPLC, electrically driven separation techniques such as CE and CEC are increasing in importance. Both techniques have already been successfully coupled to spectroscopic detection techniques and show potential for many real-life applications [10]. Overall, the on-line coupling of spectroscopy and chromatography is a prerequisite for the successful treatment of the problems encountered in genomics, proteomics, and metabolomics.

Development of an HPLC-NMR method for the determination of PAHs in soil samples – a comparison with conventional methods

“…This gain in sensitivity, developments in magnet technology and pulse sequences, more rugged solvent suppression techniques enabling gradient HPLC-NMR runs, and capillary HPLC-NMR will further improve the capability of the HPLC-NMR and increase the acceptance of the method [22,23].…”

Electrospray ionization mass spectrometry screening of piperidine alkaloids from Senna spectabilis (Fabaceae) extracts: fast identification of new constituents and co-metabolites

“…[10][11][12] Structural elucidation of natural cometabolites in complex mixtures has also been possible by use of electrospray ionization tandem mass spectrometry (ESI-MS/MS), even from small quantities of plant material or other biological samples. 13,14 The use of this technique has increased substantially nowadays, and ESI-MS/MS experiments can be useful for various studies on natural product chemistry, including biosynthesis, [15][16][17][18] metabolomic profile 19,20 and dereplication.…”

Sample optimization for rapid identification of nucleosides and bases from ascidian extracts using ESI-MS/MS

“…[14][15][16] Structure elucidation of natural compounds in complex mixtures has also been possible by use of direct injections of crude extracts into the electrospray ionization tandem mass spectrometry (ESI-MS/MS) followed by isolation into the analyzer, even using small quantities of plant material or other biological samples. 17,18 More recently, systematic investigation on ionization processes and gas phase chemistry involved in natural products fragmentation for fingerprint analysis has been useful from plants, 19,20 algae, 21 and microorganisms.…”