High-performance liquid chromatography of tyrosine-containing peptides by pre-column derivatization involving formylation followed by fluorescence reaction with 1,2-diamino-4,5-dimethoxybenzene.
A pre-column fluorescence derivatization method is described for the high-performance liquid chromatographic determination of tyrosine-containing peptides. A tyrosyl residue in the peptide is first formylated in an alkaline medium in the presence of chloroform, and the resulting aldehyde is then converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. The derivative is separated on a reversed-phase column (LiChrosorb RP-18) by isocratic elution with an aqueous acetonitrile-containing potassium chloride-hydrochloric acid buffer (pH 2.2) and sodium 1-hexanesulphonate. The method is selective and fairly sensitive; the lower limits of detection for the tyrosine-containing peptides tested are in the range 3.4-26.2 pmol in a 100-microliter injection volume.
J
J
Ishida
M
M
Kai
Y
Y
Ohkura
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006878:Hydroxides; D007700:Kinetics; D010455:Peptides / Q000302:isolation & purification*; D010655:Phenylenediamines; D011188:Potassium; D017680:Potassium Compounds; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship; D014443:Tyrosine
D006878:Hydroxides; D010455:Peptides; D010655:Phenylenediamines; D017680:Potassium Compounds; C047826:1,2-diamino-4,5-dimethoxybenzene; D014443:Tyrosine; D011188:Potassium; C029943:potassium hydroxide