Looking to the future, evolutionary development can be expected because the separating power of multidimension liquid chromatography is greatly increased over single-dimension liquid chromatography. The simultaneous improvements on both the software and the chromatographic apparatus will lead to a system capable of automatically developing analytical methods for a wide range of analytes in many different matrices. Other desirable aspects are a reasonably short analysis time and Sexible operating conditions. The thrust of multidimensional research will most probably be to improve the combination of separation methods, including coupling to alternative separation techniques.

In this paper the application of on-line HPLC–UV–APCI (atmospheric pressure chemical ionization) mass spectrometry (MS) coupling for the separation and determination of different carotenoids as well as cis/trans isomers of β-carotene is reported. All HPLC separations were carried out under RP conditions on self-synthesized polymeric C30 phases. The analysis of a carotenoid mixture containing astaxanthin, canthaxanthin, zeaxanthin, echinenone and β-carotene by HPLC–APCI-MS was achieved by scanning the mass range from m/z 200 to 700. For the characterization of a sample containing cis/trans isomers of β-carotene as well as their oxidation products, a photodiode-array UV–visible absorbance detector was used in addition between the column and the mass spectrometer for structural elucidation of the geometrical isomers. The detection limit for β-carotene in positive-ion APCI–MS was determined to be 1 pmol. In addition, an extract of non-polar substances in vegetable juice has been analyzed by HPLC–APCI-MS. The included carotenoids could be identified by their masses and their retention times.

A crude palm-oil extract rich in vitamin E homologues was investigated by HPLC-MS and HPLC-NMR coupling. For mass spectrometry a newly introduced ionization technique called Coordination Ion Spray (CIS) was used. Through the addition of silver ions to the HPLC eluent, the ionization process of nonpolar substances is facilitated. Chromatography and all coupling experiments were conducted on a C(30) column which exhibited an extraordinary shape selectivity and overwhelming sample-loading capability. Experiments were performed with pure methanol as an eluent which proved to be ideal for NMR spectroscopy as well as mass spectrometry. All necessary information for unambiguous structural assignment was collected within 45 min of the LC-NMR experiment and 15 min of the LC-MS experiment. Six compounds were identified, i.e., α-, β-, γ-, and δ-tocotrienol, α-tocoenol, and α-tocopherol.

The hyphenation of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and molecular compositions of mixtures. Most of the routinely used NMR flow-cells have detection volumes between 40–180 μL for conventional separations with analytical columns, and the newest designs employ detection volumes in the order of 200 nL for capillary separations. The low flow rates used in capillary chromatography permit the use of deuterated solvents. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow capillary HPLC-NMR experiments.

What causes the shape selectivity of C30 phases? This question can be answered by combining NMR and fluorescence spectroscopies with HPLC separations at various temperatures. The selectivities depend on the ratio of trans to gauche conformations of the alkyl chains, whose dynamic behavior was characterized with a two-dimensional solid-state NMR spectrum (shown on the right).

Worauf beruht die Selektivität von C30-Trennphasen? Diese Frage ließ sich mit einer Kombination aus temperaturabhängigen NMR- und Fluoreszenzmessungen sowie temperaturabhängigen HPLC-Trennungen beantworten. Die Selektivität basiert auf dem Verhältnis von trans- zu gauche-Konformationen der Alkylketten, deren dynamisches Verhalten durch ein 2D-Festkörper-NMR-Spektrum (siehe rechts) ermittelt werden konnte.