Precolumn Derivatization of Peptides with Phenylisothiocyanate (PITC)
A rapid and ultra sensitive method for the purification and quantitation of peptide hormones is described. The method is based on pre column derivatizatiori of peptides with phenylisothiocyanate (PITC) to form the phenylthiocarbamoyl derivatives (PTC-peptides). The derivatized peptides are analyzed by reverse-phase high performance liquid chromatography (HPLC) on a Zorbax ODS column and detected at 269 nm at a limit sensitivity of 1 picomole. The eluted material can be either analysed for amino acid composition or structural sequence, using available microanalytic techniques. The HPLC analysis of PTC-dynorphin-(1–13), PTC-dynorphin-(1–11) and PTC-Leu-enkephalin allowed the complete seraration of each peptide with high recovery (>75%). The method can be used at the final purification step of naturally-occur ring peptides and for the evaluation of the purity and amount of material than can be submitted either to amino acid analysis or to Edman degradation.
KeywordsHigh Performance Liquid Chromatography Acetonitrile Pyridine MeOH Isothiocyanate
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