High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives.

Publisher Summary High-performance liquid chromatography (HPLC) provides a rapid, sensitive, and reproducible means of separating and quantifying simultaneously a variety of sulfur-containing amino acids and related derivatives. The HPLC method described in this chapter is modified and is based on the initial formation of S-carboxy-methyl derivatives of free thiols followed by the conversion of free amino groups to 2,4-dinitrophenyl (DNP) derivatives. Following derivatization, nanomole levels of individual sulfur-containing amino acids are measured using UV detection at 365 nm after separation by reverse-phase ion-exchange HPLC. Because of the versatility of this HPLC method, biological specimen preparation as well as derivatization and HPLC analysis procedures are discussed. DNP derivatives of acidic amino acids (including thiol-containing compounds) are separated on a 3-aminopropyl column by reversed-phase ion-exchange HPLC. In the mobile phase, methanol is used to elute rapidly the excess 2,4-dinitrophenol and the DNP derivatives of basic and neutral amino acids. Acetic acid is present in the mobile phase to maintain the bonded-phase amino groups in the protonated form. By increasing the sodium acetate concentration of the mobile phase, selective elution of acidic DNP derivatives is accomplished. The eluted DNP derivatives are measured by detection at 365 nm.