Selective fluorescence derivatization and capillary electrophoretic separation of amidated amino acids

Selectively derivatized amide-terminated amino acids were separated by mice llar electrokinetic capillary chromatography (MECC). The amides were select ively derivatized by deactivating all primary amines in the sample mixture by acetylation, converting the amides to primary amines by Hofmann rearrang ement, and tagging the resultant amines with fluorescein isothiocyanate (FI TC). The fluorescent amide derivatives were detected by confocal laser-indu ced fluorescence. The use of the MECC mode was mandated by very similar cha rge and size characteristics of the derivatized amides. Separation of 11 am ino acid amides was carried out in bare fused silica capillaries in 10 mM b erate and 90 mM sodium dodecyl sulfate buffer in under 15 min. Analysis of dispersion and mobility behavior suggests that hydrophobicity is the primar y determinant of micelle/buffer partitioning, and, therefore, mobility; rel ative hydrophobicity based on values for amino acids is an adequate predict or of elution order for derivatives without charged side-chains. Selectivit y of the reaction was estimated to be as great as 100, perhaps greater; the estimation was limited by ability of FITC to derivatize at low concentrati ons. Rearrangement reaction yields and kinetics were found by NMR spectrosc opy to depend on side-chain identity. Yields were 80-100% for the rearrange ment, and half-lives were 10-30 min for 100 mM solutions of several represe ntative amino acid amides. Reaction products were not purified, for the sak e of simplicity, and a number of impurity peaks appeared in the electropher ograms. Because of the high efficiency of the separation (optimal plate hei ghts below 2 mu m), resolution was usually adequate to mitigate this potent ial problem. (C) 1999 Elsevier Science B.V. All rights reserved.