Separation of amino acids as derivatives of phenylthiohydantoin (PTEkmino acids) has been performed by paper and thin-layer chromatography, gas chromatography and high-performance liquid chromatography (HPLC). EIPLC, being a rapid and highly
efficient technique, is advantageous because all PTH-amino acids (even non-volatile ones) can be determined as such. Up till now, separations have required several different eluent systems1A or gradient eIution2~5-11; reversed-phase systems with bonded phases have been used most frequently. The use of isocratic elution, as proposed by Zimmermann et aZ.l' should lead to simple and inexpensive analytical systems, especially with a one-column design. Fong and Grushka13 used a bonded tripeptide phase, giving unique retention orders, but low-efficiency chromatograms.
In this paper we describe an efficient isocratic reversed-phase system with which 21 PTH-amino acids out of the 22 normally encouutered in the Eciman degradation of peptides and proteins can be separated. Increased column temperature is a prerequisite for the separation. EXPERIMENTAL Reagents The eiuent was a mixture of 25 parts (v/v) of acetouitrile (Merck, UVASOL, or Rathbum Chemicals, HPLC grade S) with 75 parts of 0.01 M sodium acetate; the pII of the mixture was adjusted to 4.6 with concentrated acetic acid. PTH-amino acids were obtained from Pierce and from Sigma; stock solutions in methanol (Rathbum Chemicals) or a mixture of methanol and ethyl acetate (Merck) were CQ. 20 m&f.
Apparatus and chromatograptric tecfuz fque
The column consisted of two stainless-steel-tubes (each 15 cm x 3.9 mm I.D.)