Sabine Strohschein

Separation and identification of various carotenoids by C30 reversed-phase high-performance liquid chromatography coupled to UV and atmospheric pressure chemical ionization mass spectrometric detection

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 beta-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 beta-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 beta-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 beta-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.

Separation and Identification of Tocotrienol Isomers by HPLC−MS and HPLC−NMR Coupling

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.

Capillary HPLC−NMR Coupling: High-Resolution 1H NMR Spectroscopy in the Nanoliter Scale

Coupling HPLC and NMR is one of the most powerful techniques for simultaneous separation and structural elucidation of unknown compounds in mixtures. To date, however, minimizing the detection volume, as is required when coupling NMR with miniaturized separation techniques, has been accompanied by a dramatic loss in resolution of the NMR spectra. Here, we report on the coupling of gradient capillary HPLC with on-column, high-resolution NMR detection. On-line stopped-flow and static (1)H NMR spectra were acquired with capillary columns of 75-315 μm i.d. With detection over a length of 1.2 cm, cell volumes cover a range of 50-900 nL. An on-line-detected NMR separation of dansylated amino acids was carried out in a 315 μm i.d. fused silica capillary packed to a length of 12 cm with C(18) stationary phase. The low solvent consumption makes the use of fully deuterated solvents economically feasible. NMR spectra with resolution on the order of 3 Hz were obtained using a 50 nL detection cell to measure 1.1 nmol of dansylated γ-aminobutyric acid under static conditions in a 75 μm i.d. capillary.

On-line coupling of separation techniques to NMR

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.

On-line trace enrichment in hyphenated liquid chromatography-nuclear magnetic resonance spectroscopy.

Abstract The potential of on-line trace enrichment combined with column liquid chromatography–nuclear magnetic resonance detection (LC–NMR) is demonstrated for the identification of hop bitter acids in hop extracts. By performing on-line solid-phase extraction on 1–5 ml of hop extract (which contains the six major hop bitter acids) the detection/analysis time in the stopped-flow detection mode could be reduced 4-fold, from 60 min to 15 min per individual peak. The applicability of combined trace enrichment and separation using a single short (12.5 mm×4.6 mm I.D.) high-pressure-packed column combined on-line with NMR was also studied. Sample enrichment of 5 ml of a solution spiked with three drugs at 5 μg/ml was sufficient for the acquisition of high-quality spectra both in the stopped-flow and even in the continuous-flow detection mode. The examples demonstrate that analyte detectability can be significantly improved by performing on-line sample enrichment prior to LC–NMR analysis.

Comparison of the separation of cis/trans isomers of tretinoin with different stationary phases by liquid chromatography-nuclear magnetic resonance coupling

Abstract The comparison of two different types of RP stationary phases in their ability to separate cis/trans isomers of retinoic acid (tretinoin) has been investigated by LC-NMR coupling. Only by recording of 1 H NMR spectra the structural identification of the separated compounds was possible, since their absorption coefficients are very similar and their mass is identical, and therefore identification by UV-Vis is not unambiguous and identification with LC-MS fails due to identical fragmentation patterns. A commonly used C 18 phase and a recently developed C 30 phase have been used for the separation of a mixture of thermal isomerized retinoic acids. Three isomers could be separated and identified with the separation on a C 18 column, whereas five cis/trans isomers could be identified by the use of a C 30 column. It could be shown that even under optimized chromatographic conditions the separation efficiency of the two phases varies and that these differences are probably due to differences in the alkyl chain organization of the stationary phases.

Separation and identification of vitamin A acetate isomers by supercritical fluid chromatography—1H NMR coupling

Abstract Unlike HPLC-NMR coupling, the combination of supercritical fluid chromatography (SFC) with NMR spectroscopy is not yet a routine method, but the feasibility of this coupling technique has already been demonstrated with synthetic mixtures. In this paper the first real life application is presented and the potential of this new method is shown. A mixture of five cis/trans isomers of vitamin A acetate is separated by SFC using supercritical CO 2 as eluent. Therefore no solvent signal suppression is necessary and the unrestricted observation of the whole spectral range is possible, contrary to the HPLC separation in n -heptane, where in the aliphatic region almost 2 ppm of the 1 H NMR spectrum are affected by the suppression technique.

Hyphenation of high performance liquid chromatography with nuclear magnetic resonance spectroscopy for the characterization of β-carotene isomers employing a C30 stationary phase

The hyphenation of HPLC together with NMR spectroscopy proves advantageous for the structure elucidation of oxidation- and UV-sensitive compounds such as beta-carotene isomers. In the closed-loop HPLC-NMR system, degradation or isomerization of separated compounds is largely hindered. With the help of 3-microm C30 stationary phases a better separation efficiency towards the different beta-carotene cis/trans isomers could be obtained in comparison to a 5-microm material, resulting in sharper peaks and a better resolution of all compounds. This effect greatly facilitated the structure determination of the isomers by HPLC-NMR coupling. Due to the introduction of a superior stationary phase, the structure of seven cis-isomers of beta-carotene could thereby be determined employing the stopped-flow HPLC-1H-NMR mode.

CONFORMATIONAL ASPECTS OF THE P-NITROSOPHENOLATE ANION AND RELATED COMPOUNDS : NMR STUDY AND AB INITIO 6-31G OPTIMIZATIONS OF MOLECULAR STRUCTURES

Abstract The p-nitrosophenolate anion, PNPA (3), represents an excellent example of variable π-electron transfer demanded by a network of different hydrogen bonds. The limiting structures of mono-protonation in the solid state and in solution are either the p-nitrosophenol (1) or the p-quinonoxime (2) molecules. For the systems 1 to 3 and the related compounds 4 to 7 are results of ab initio 6-31G HF MO optimizations of molecular structures presented which are calculated additionally in variously rotated conformations. Solid state 13C-CP/MAS NMR spectra of two PNPA salts 8 and 9 have been recorded at 7.0 Tesla. The protonation of 8 and 9 was studied in solution using a 400 MHz NMR spectrometer recording the variations in 1H and 13C NMR chemical shifts on changing the solvent from DMSO-d6 to D2O in steps of 25%. The 2D 1H-13C correlated spectrum leads to unambigous assignment of NMR signals. From the coalescence temperature of 1H signals of 9 the experimental barrier of rotation in D2O was determined as 16.9 ± 0.1 kcal mol-1. The calculated gas phase rotational barrier for free PNPA is with 26.9 kcal mol-1 much higher, but is lowered by calculations for the Li-salt 6 c to 14.5 kcal mol-1 and to 11.8 kcal mol-1 for the protonated PNP If. The in-plane inversion of PNPA through 3c is calculated to be extremely high with 52.4 kcal mol-1. The effects of rotation of substituents to orthogonal conformations on calculated energies and on intramolecular distances were studied thoroughly.