Jan 1, 1984

Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate

Analytical Biochemistry
R L Heinrikson, S C Meredith

Abstract

Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable ...Continue Reading

  • References1
  • Citations529

References

  • References1
  • Citations529

Citations

Mentioned in this Paper

Amino acid derivative
Derivatives
Amino Acids, I.V. solution additive
Isothiocyanates
Proline
High Pressure Liquid Chromatography Procedure
Pancreatic ribonuclease
Ammonium acetate
Polypeptides
Leftose

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