This document describes the optimization of peptide separations using PLRP-S reversed-phase material in acidic and basic conditions by HPLC with ELSD.
The document details a method for separating angiotensins, a peptide hormone mixture, utilizing PLRP-S reversed-phase material which is stable across a wide pH range (1-14). This stability allows the use of both conventional acidic ion pairing agents like trifluoroacetic acid (TFA) and alkaline conditions to optimize selectivity based on amino acid charge distribution.
The study compares separation results using different mobile phase modifiers. Under acidic conditions, varying concentrations of TFA were tested, showing that while 0.1% provides good peak shape, it causes co-elution of Angiotensin II and III. Reducing the concentration improves resolution but can cause band broadening. Alternatively, under basic conditions, ammonium hydroxide was used as a modifier to achieve separation.
The procedure uses a constant flow rate of 1.0 mL/min.
The separation takes 15 minutes to complete using a linear gradient from 10% to 100% B.
The detector settings for Figure 1 require a neb temperature of 85 °C and an evap temperature of 75 °C.