The enantiomeric resolution of several dipeptides, amino acid (i.e., isoleucine) and tripeptide (i.e., Leu‐Gly‐Phe) with two stereogenic centers on β‐cyclodextrin bonded chiral stationary phase (β‐CD CSP) using polar‐organic acetonitrile as the mobile phase is examined through pre‐column chemical derivatization with a series of tagging reagents such as benzoyl chloride, benzenesulfonyl chloride and 1‐naphthalenesulfonyl chloride. These tagging reagents are similar in structure; however, the enantioselectivity for the same analyte derivatized with these tagging reagents is quite different and found to be the best with benzoyl chloride. In the reversed‐phase mode or on the γ‐CD CSP under the same chromatographic conditions, the enantioresolution diminishes for all tagged enantiomers that were examined in this study. Dipeptides derivatized by benzoyl chloride appear to be better resolved than by dansyl chloride as reported previously. Interestingly, no enantioresolution for most derivatized amino acids with single stereogenic center was observed. Finally, enantioresolution can be enhanced by replacing the basic additive such as triethylamine with tripropylamine in the polar‐organic mobile phase.
A HPLC system linked to a variable wavelengths UV detector was used to sequentially determine the total concentration and enantiomeric composition of two selected free amino acids (leucine, phenylalanine) in two highly fermented foods, bean curd and paste soy bean (i.e., miso), after their derivatization with N‐benzoyl chloride. As was expected, fermented bean curds tended to have a higher percentage of D‐enantiomers and greater total amount of amino acids than paste soy beans or fermented soy beans as reported previously. In one case, the relative amounts of D‐phenylalanine and D‐leucine exceeded 40% of the individual amino acids. Interestingly, paste soy bean made in Japan is found to be better than that made in Taiwan in terms of a higher total level of amino acids and a lower percentage of D‐enantiomers. In most samples, leucine was found to have a lower average percentage of D‐enantiomer but higher total concentration than phenylalanine. This finding is consistent with what was observed for fermented soy beans. Only a trace of amino acids (leucine, phenylalanine) was found in raw soybeans and bean curd, which was thought to be a result of a lack of microbial activities (e.g., fermentation, aging, etc.).
The enantioresolution of thirteen methylthiohydantoin‐amino acids (MTH‐amino acids), which are substrates for producing D‐amino acids through enzyme‐catalyzed hydrolysis, is described on R, S‐2‐hydroxypropyl derivatized β‐cyclodextrin bonded stationary phase (RSP‐β‐CD CSP) with a water‐based mobile phase. The enantioresolution is relatively sensitive to the structural variation of group which is attached to the carbon at position five on methylthiohydantoin moiety and subsequently turns into side‐chain group of corresponding D‐amino acid produced after hydrolysis. The inclusion complexation is believed to be the mechanism responsible for the observed enantioresolution that cannot be reproduced either on native β‐cyclodextrin CSP under the same chromatographic conditions or on both CSPs using the acetonitrile‐based mobile phase. Approaches for enantioimprovement include varying the percentage of organic modifier in the mobile phase and using the buffer‐typed mobile phase such as triethylammonium acetate (pH 4.1).
A HPLC approach using R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin packed column as the stationary phase was developed to resolve five nucleic‐acid bases and an a log hypoxanthine in the reversed‐phase mode. These bases are not only similar in structure but also very close in basicity. However, the resolution can be completed in less than ten minutes and is considered to be better carried out on the R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin phase than that obtained on the native β‐cyclodextrin phase under the same chromatographic conditions. The mechanism involved in the resolution is believed to be inclusion complexation between the analyte and the cavity of cyclodextrin in the reversed‐phase mode. The retention time was found relevant to the size of the analyte. The number of groups on analyte that is available to form hydrogen bonding with hydroxyl groups on CDs also affects the retention scale. Factors of introducing organic acid and base or organic modifier such as methanol to the water‐based mobile phase or increasing their percent ages in the mobile phase decreases the retention time without de grading the resolution significantly.
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