Separation of lissamine rhodamine B sulfonyl derivative of amino acids by high-performance liquid chromatography and thin-layer chromatography

Abstract

The effectiveness of laryl chloride (Lissamine rhodamine B sulfonyl chloride) as a reagent for labeling free amino groups of amino acids, peptides and proteins has been demonstrated. Laryl amino acids are bright red compounds which absorb light with a maximum at a wavelength of 560 nm and emit light with a maximum at a wavelength of 595 nm. On thin-layer plates, fluorescence is about 125 times more sensitive than visual observation of the colored spots and it was possible to observe laryl amino acids in amounts as low as 400 fmol, about half the amount which is needed for dansyl amino acids. The colorimetric thin-layer methods are as sensitive as the dabsyl chloride method and do not require development with hydrochloric acid fumes. A high-performance liquid chromatographic method was developed for the separation of the laryl amino acids and the usefulness of the technique was demonstrated on small peptides.