Extinction Coefficient

Firstly, free amino acid solution of protein sample was obtained by using hydrolysis in 6M HCl., The pre-column derivatization of amino acids was performed to obtain the absorbent group and hydrophobic structure, then reversed-phase liquid chromatography with UV detector was used for separation and detection. Phenyl isothiocyanate (PITC) derivatization: amino acidscan react with PITC under mild alkaline conditions (pH≈9.0) and generate thiazolinone aniline derivatives.  The derivatives have strong reservations in the reversed-phase column and clear absorption under 254 nm UV.

Methods:Ultraviolet absorbance assay at 280 nm (A280) → Acid hydrolysis → Amino Acid Standard Preparation → Phenylisothiocyanate (PITC) derivatization → HPLC analysis → Data processing