An Improved Injection System for On-line High Sensitivity Phenylthiohydantoin Amino Acid Analysis

Abstract

The first commercial protein sequencers introduced in 1970 and the more recent “gas-phase” sequencer (Hewick et al., 1981) failed to appreciate the extent of heat destruction of certain derivitized amino acids while sitting in the fraction collector awaiting manual analysis. This problem was largely overcome by on-line PTH-amino acid analysis (Machleidt et al., 1980). In spite of the advantages of on-line PTH-amino acid analysis several shortcomings still exist in the commercial sequencers (Applied Biosystems Inc., models 470A and 477A equipped with model 120A PTH-amino acid analyser). The percentage of sample injected onto the HPLC column is <50% while the remainder is transferred to a non-refrigerated fraction collector where labile PTH-amino acids decrease due to destruction. A second difficulty is maintaining parameters (flow rate, time) which control the transfer of liquid from the conversion flask of the sequencer into the sample loop of the HPLC injection valve.