Enantioseparations of FMOC-amino acids Using Polysaccharide based Chiral Stationary Phases Under Polar Organic Mobile Phase Conditions

Author: Ana Gogolashvili
Keywords: Liquid Chromatography, Enantiomers, Chiral Analysis, FMOC-Amino acids
Annotation:

N-Fluorenylmethoxycarbonyl (FMOC) -amino acids are important building blocks for the solid phase synthesis of peptides. After the development of FMOC/tBu strategy for solid phase peptide syntheses, FMOC -amino acids have become the raw materials of choice for the preparation of synthetic peptides. Using this methodology, peptides can be prepared in a few days with high yield using a peptide synthesizer. As the number of amino acids residues increases the final purity and overall yield of the peptide produced is directly affected by the chemical and enantiomeric purity of the protected amino acids used. Currently, for standard protected -amino acids, the expected enantiomeric purity is > 99% ee and sometimes even > 99.8% ee. This level of precision in measurement of enantiomeric purity can only be achieved by very few analytical techniques, chiral HPLC being one of them. The main advantages of chiral HPLC analysis over other techniques are speed (run less than 20 min), detection level and ease of use. In this study, the separation of the 19 standards FMOC protected -amino acids under polar organic separation mode using polysaccharide based chiral stationary phases is reported. As the mobile phase alcohols (methanol and ethanol) and acetonitrile with 0.1% (v/v) additive of formic acid were used. Five different polysaccharide-based chiral columns of Lux series, such as Lux Cellulose-1, Lux Celluose-2, lux Cellulose-3, Lux Cellulose-4 and Lux Amylose-2 from Phenomenex (Torrance, CA, USA) were used. The separations were performed using Agilent 1260 HPLC instrument equipped with a binary pump and variable wavelength UV-detector. In alcohols as mobile phases the best results were obtained on Lux Cellulose-1 and Lux Cellulose-4 columns. In particular, the enantiomers of almost all FMOC-amino acids were separated on these columns. Few examples of enantiomer elution order reversal depending on the chiral selector and the mobile phase were observed.

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FMOC-ამინომჟავების ენანტიომერების ანალიზი პოლისაქარიდული ქირალური სელექტორებისა და პოლარულ-ორგანული მოძრავი ფაზების გამოყენებით [ka]