Tobias Glaser

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.

Qualitative and Quantitative Determination of Carotenoid Stereoisomers in a Variety of Spinach Samples by Use of MSPD Before HPLC-UV, HPLC- APCI-MS, and HPLC-NMR On-Line Coupling

SummaryThe carotenoids lutein and zeaxanthin have been identified as the macular 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 solid-phase 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.

Synthesis and Separation of Structural Isomers of 2(3),9(10),16(17),23(24)‐Tetrasubstituted Phthalocyanines

The 2(3),9(10),16(17),23(24)-tetrasubstituted metalphthalocyanines 1-7 (M = In, Ni, Zn) were synthesized, as mixtures of four different structural isomers, from the corresponding 4-alkoxy-1,2-dicyanobenzenes and the appropriate metal salts. Separation of the four structural isomers was successfully achieved on a C30 alkyl phase by high-performance liquid chromatography (HPLC). The determination of the point groups of the structural isomers was carried out for 1 and 3, the composition of the structural isomers of 4-7 was accomplished by comparing their retention times and UV/Vis spectra with the data of 1 and 3. For the phthalocyanines 8-10 and the naphthalocyanines 11 and 12 only the C4h and D2h isomers could be separated.

Specific production of γ-polypodatetraene or 17-isodammara-20(21),24-diene by squalene–hopene cyclase mutant

Amino acids lining the catalytic cavity of squalene–hopene cyclase of Alicyclobacillus acidocaldarius were mutated to investigate their catalytic functions. Mutagenesis of Leu607 to Lys in the central part of the cavity resulted in the production of the bicyclic γ-polypodatetraene (1) as main product, while the mutation of Phe605 to Lys near the deprotonation site of the cavity led mainly to the formation of tetracyclic 17-isodammara-20(21),24-diene (2).

Application of on-line C30 RP-HPLC-NMR for the analysis of flavonoids from leaf extract of Maytenus aquifolium.

Universidade Estadual Paulista Instituto de Quimica de Araraquara, CP 355, 14801-970, Araraquara, SP

Identification of trifluoperazine metabolites using HPLC–NMR of test mixtures

The metabolites of the antipsychotic drug trifluoperazine (TFP) were investigated with on‐line coupling of high‐performance liquid chromatography (HPLC) and NMR spectroscopy. A chromatographic method was developed using a reversed‐phase C30 silica gel column. Rats were given a cumulative dose of 90 mg kg−1 TFP dihydrochloride and samples of brain and muscle tissues were extracted after 29 h. The peaks obtained on chromatographic separation were assigned to trifluoperazine and its metabolites. With the help of chromatograms obtained from test mixtures, 1D and 2D HPLC–NMR were used to identify the peaks found for these mixtures. Copyright

Unequivocal structural assignment of steroids in galenic emulsions employing HPLC‐nMR coupling

The structural identity of two steroidal components in the low μg range of a pharmaceutical gel was determined by a rapid and efficient analytical method employing online HPLC-NMR coupling. The chromatographic separation conditions were optimised towards the requirements of on-line 1 H NMR detection, employing a tailored C 30 column together with a gradient elution of a methanol/D 2 O mixture. Stopped-flow 1D and 2D 1 H NMR spectra of the investigated chromatographic peaks unequivocally revealed the structure of the corresponding steroids norethisterone acetate and estradiol.