All the organic soluble amino acid phenylthiohydantoins are eluted (but not totally resolved) in under 40 min on either a Zorbax ODS or Permaphase ETH column. The superior resolution of the Zorbax ODS column and the complimentary characteristic of the Permaphase ETH column allow the isocratic analysis of 15 phenylthiohydantoins in less than 20 min when the columns are operated simultaneously. The water soluble PTH-His and -Arg are analyzed separately in under 10 min with the Zorbax ODS column. Five picomoles of a derivative may be detected with a sample to noise ratio of 6. HPLC is the most sensitive, practical method for PTH analysis.
Most laboratories involved in the important problem of determining the amino acid sequences of polypeptides and proteins use the now classical Edman method (1). Analysis of the phenylthiohydantoins (PTHs) formed in this procedure is a major undertaking, particularly with the advent of Edman's automated procedure (2). Several methods for PTH analysis are currently in use but most popular are thin-layer chromatography (TLC) (3), gas chromatography (GC) (4), and back hydrolysis (hydrolysis of the PTHs and analysis of the recovered amino acids) (5).
Recently, we reported on the analysis of PTHs by high performance liquid chromatography (HPLC) (6-g), a technique which is more sensitive than GC and applicable to the analysis of all the PTHs (including PTH-Arg) without derivatization.
Unfortunately, some PTHs were not separated. We now report better resolution and higher sensitivity achieved by the use of new column packings. The new technology has been applied to the analysis of PTH samples from automated Edman degradations.
METHODS AND MATERIALS
Analyses were performed with a DuPont Model 830 Liquid Chromatograph equipped with a gradient elution device and uv monitor with 254 nm emission (Instrument Products Div.