Hydrolysis of samples is performed in a PicoTag Workstation. Amino acids are derivatized and separated on an Agilent 1260 liquid chromatograph with” Chemstation” software that controls the LC and collects, analyzes and reports the data. The G1367E autosampler performs pre-column derivatization and multiple sample handling.
Derivatized amino acids are eluted from a narrow bore, (2.1 x 200 mm), (Hypersil AA-ODS), 5 um reverse phase column purchased from Thermo Fisher (part # 30105-202130). Solvent A consists of a 20mM Na acetate buffer with 0.018% v/v triethylamine (Fluka 90338), 0.05mM EDTA, (Sigma E4884) and 0.3% tetrahydrofuran (Fluka 87363) adjusted to pH 7.2 with weak acetic acid. Solvent B is a 20% 100 mM Na acetate buffer (pH 7.2) with 40% acetonitrile and 40% methanol. The working gradient begins at 0 minutes at 100% A at 0.45 ml/min and goes to 60% B over 17 minutes.
Primary amino acids (tagged with OPA, Agilent Item # 5061-3335) are detected at 338/390 nm by the Variable Wavelength (UV) detector (G1365D) and secondary amino acids (tagged with FMOC, Agilent Item #5061-3337 ) at 266/324 nm.
Our flourometric detector (G1321B) monitors the primaries at excitation/emission 340/450 and the secondaries at 266/305.