Highly selective reversed phases (C(30) phases) are self-packed in 250 microm inner diameter fused-silica capillaries and employed for capillary HPLC separation of shape-constrained natural compounds (tocopherol homologues, vitamin E). Miniaturized hyphenated systems such as capillary HPLC-ESI-MS (positive ionization mode) and, with special emphasis, continuous-flow capillary HPLC- NMR are used for structural determination of the separated compounds. Despite the small amount of sample available (1.33 microg of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow (1)H NMR conditions, thus allowing an immediate peak identification. Further structural assignment was carried out in the stopped-flow NMR mode as shown, for example, by a 2D (1)H,(1)H COSY NMR spectrum of alpha-tocopherol. We demonstrate in this paper the considerable potential of hyphenated capillary separations coupled to MS and NMR for the investigation of restricted amounts of sample.

SummaryThe carotenoids Jutein and zeaxanthin have been identified as the macuJar pigments of the human retina. Nutritional epidemiological reports indicate that high consumption of fruits and vegetables rich in these carotenoids is correlated with reduced risk of some illnesses, e.g. agerelated macular degeneration (AMD).Because carotenoids are extremely sensitive to UV light and oxygen, they occur in nature as several Z/E stereoisomers which can differ considerably in their biological effectiveness. With particular regard to dietary supplementation we have focussed on identification and quantification of all the carotenoid stereoisomers occurring in a variety of raw and processed homegrown and commercial spinach samples, a natural source of carotenoids. Isolation of the unstable carotenoid stereoisomers from biological tissues without sample-preparation artifacts requires a mild, rapid, complete, and reproducible extraction technique such as matrix solidphase dispersion (MSPD). Separation and unequivocal structural elucidation of the main carotenoid stereoisomers was achieved by use of hyphenated analytical techniques and exclusion of light and oxygen. HPLC analysis with highly selective C30 columns was used for quantitative determination of the main Z/E carotenoid stereoisomers and HPLC-APCI-MS and HPLC-NMR on-line coupling was used for unequivocal structural elucidation. Whereas HPLC-APCI-MS can distinguish between the carotenoids lutein and zeaxanthin, HPLC-NMR enables identification of all the main Z/E stereoisomers.

Gingival samples treated with toothpastes containing tocopherols (vitamin E) were investigated employing HPLC chromatography. The aim was to verify that vitamin E is actually enriched in the tissue, which could have beneficial effects on oral health. After determination of the tocopherols available in the toothpastes, control samples from healthy test persons and subjects suffering from gingivitis were analyzed. Subsequently, gingival tissues from diseased test persons who treated their teeth with the toothpastes containing tocopherols using various kinds of concentrations or applications were investigated. The first step of the analysis was a fast and careful extraction employing matrix solid-phase dispersion (MSPD). Afterward, the separation of the different tocopherol homologues existing was performed by HPLC chromatography on highly selective C30 RP phases. The identification of the tocopherol homologues was performed using the on-line coupling of HPLC with NMR spectroscopy and mass spectrometry.

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.

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