The model validation is based on the mean hold-up determination upon a representative column compartment, which is compared to experimental data.
The peptide samples (10 20 µg[LC2]) were separated on an Ascentis Express Peptide ES-C18 column (2.7 μm particle size, 160 Å pore size, 5 cm length × 2.1 mm i.d., coupled to a 5 mm × 2.1 mm i.d. guard column with similar particle and pore size; Sigma-Aldrich, St . Louis MO, USA), with the system operating at a flow rate of 0.400 mL/min and column compartment at 60 °C.
When a flow cell was in direct thermal contact (metal on metal) with the detector optical bench, the mobile phase temperature was actively controlled to the measured flow cell temperature, by using one of the heat exchangers (HX) in the column compartment.
The autosampler and column compartment were maintained at 25°C and 35°C, respectively.
The column was maintained at 40°C by the column compartment.
Agilent 1200 series system consists of G1311A binary pump, G1322A degasser, G1367B HIP-ALS autosampler, and G1316A thermostatted column compartment.
Chromatographic separation was carried out on HPLC instrument (Agilent 1260 Infinity, Germany) equipped with a G1361A pump, G1316A thermo-stated column compartment, and G2260A auto-sampler.
HPLC was performed using an UltiMate 3000 liquid chromatographic system (Thermo-Dionex Corporation, USA) equipped with a DAD, an auto sampler, and a column compartment.
The chromatographic system used was an Agilent-1100 series (Bangalore, India) comprised of degasser, quaternary pump, auto injector, column compartment and photodiode array detector.
An Agilent 1290 Infinity HPLC system (Agilent, Santa Clara, CA, USA) was equipped with a controller, two pumps, a column compartment, and a degasser.
Semipreparative HPLC was carried out on an Agilent 1200 series instrument equipped with a quaternary pump, a vacuum degasser, an autosampler, a thermostatic column compartment, and a diode array detector.
