Fundamentals of Oligonucleotide Separation by HPLC

Per- and polyfluoroalkyl substances (PFAS) are exceptionally versatile chemicals and have seemingly endless applications. Due to their persistent nature; prevalence in blood, food and environment; and emerging toxicity, there is a significant concern for their presence in the human body. Analysis is especially complicated due to background contamination in laboratory supplies and system. This paper will discuss sample preparation and LC-MS/MS analysis of an expanded panel of PFAS compounds in serum.

Oligonucleotides—therapeutic agents for difficult-to-treat diseases—rely on precise molecular weight confirmation to ensure quality during development. Experts from Agilent Technologies explore the challenges of liquid chromatography-mass spectrometry (LC/MS) analysis in oligonucleotide synthesis, as well as strategies for accurate deconvolution, method development, and optimal equipment selection.

This application note examines the selectivity differences between three L1 columns. The CORTECS Premier C18, CORTECS Premier C18+ and CORTECS Premier T3 Columns were used to separated paracetamol and its impurities using EP monograph conditions.

In this application note, a USP monograph is modernized to a shorter column using 5 µm particles, which can be allowable under USP <621> as long as the N value is within the guidelines. By using highly efficient CORTECS 5 μm particles, this type of modernization is possible and reductions in solvent usage and run times are achieved.

This non-IP-RP method, using ammonium bicarbonate as mobile phase additive, is highly sensitive and suitable for the bioanalysis of therapeutic oligonucleotides.

In this work the development of two key attributes of the 5um CORTECS Columns is examined. First column efficiency is compared across CORTECS and other solid-core 5 μm columns. Next scalability from sub-2 μm to 5 μm particles is examined between the CORTECS Column lines and competitive column lines. It was found that CORTECS columns have higher efficiency compared to other solid-core columns and that CORTECS particles are fully scalable.