Impact of HPLC Pump Performance and Instrumentation on Non-Specific Adsorption of Peptides

With the increase in available biotherapeutics, peptide mapping is increasingly used for quality control as it is used for characterization and impurity testing of these complex biomolecules. These analyses require demanding method conditions to achieve acceptable chromatographic separation, including low flow rates and long, shallow gradients. These are challenging conditions to produce repeatably for many high and ultra-high performance liquid chromatography (HPLC/UHPLC) pumps. As a result, accurate and precise pump performance is critical to success with these types of reversed-phase protein digest separation methods. Furthermore, transferring across different systems can create unique challenges.

Aside from challenges to achieving repeatable separation, the wide range of chemical properties of the peptides pose unique challenges. Some acidic and phosphorylated peptides are metal sensitive and may interact with a standard stainless steel flow path potentially resulting in decreased recovery, poor area precision, and increased peak tailing. Common strategies to mitigate non-specific adsorption include the use of mobile phase additives, chemical passivation, or sample priming. However, these solutions can be time consuming and unreliable. For this reason, a variety of technologies have been developed to reduce non-specific adsorption.

In this study, an enolase digestion standard is used as a representative complex sample across HPLC systems designed for bio related applications. The analysis was performed using typical reversed phase method conditions. The performance of a wide range of systems was evaluated with comparisons based on chromatographic criteria, including area recovery, sensitivity, and peak asymmetry/tailing. Recent developments in HPLC design allowing for reduced non-specific adsorption will be evaluated to assess impact based on analyte properties.