amino acids of interest in automated and manual sequencing in one embodiment using automated instruments manufactured by applied biosystems gradient modification is achieved by changing the manufacturer supplied gradient program which controls the operation of the hplc pumps in the instrument using the methods of the present invention the correct and reliable assignment of tryptophan residues is possible and was reproducible over time even with small sample sizes application_number US 49306290 A description sequence analysis was performed on an applied biosystems model 477a protein sequencer equipped with an applied biosystems model 120a on line pth amino acid analyzer all operations using the applied biosystems instrument were carried out as prescribed in the manufacturer 39 s user manual a pth c18 reverse phase column applied biosystems part 0711 0203 was used for separation of pth amino acid derivatives chromatography solutions were as follows solution a was made by mixing 22 ml 3m sodium acetate ph 3 8 applied biosystems 400319 6 ml 3m sodium acetate ph 4 6 applied biosystems 400199 with 500ul 12 5 v v trimethylamine and 1000 ml 5 v v tetrahydrofuran solution b consisted of acetonitrile containing 500 nmol 1 of dmptu applied biosystems 400349 unless otherwise stated all solvents were analytical or spectroscopic grade in disclosed embodiments the column was equilibrated for 12 5 minutes with solution at a concentration of 11 solution b by volume chromatography was performed at 50 55 u00b0 c preferably at 55.