Experimental determination and observations of molecular diffusion coefficients of oligonucleotides via the Taylor-Aris method.

Oligonucleotides (ONs) are a rapidly emerging class of therapeutics, with the potential to treat a wide range of diseases by targeting RNA and DNA sequences with high specificity. Their complex structures and manufacturing processes generate closely related impurities that must be meticulously monitored, necessitating techniques such as High-Performance Liquid Chromatography (HPLC). However, the analysis of ONs remains highly challenging, and a deeper understanding of ON separations and efficient column design require identifying all factors contributing to band broadening, a task currently hindered by limited insight into column performance. A key parameter influencing ON separation performance is the molecular diffusion coefficient (Dm). This study investigates the applicability of the Taylor-Aris (TA) method to accurately measure Dm values for ONs. Therefore, a comprehensive workflow is established, starting with an estimation of the theoretical Dm value of ONs based on Young's equation, to define an initial transitional flow rate to execute TA experiments. The applied flow rate is subsequently optimized, based on observed discrepancies between theoretical and experimental transitional flow rates to ensure accurate Dm determination. Measurements are conducted for homo-oligonucleotides with varying chain lengths (T5, T10, T20, T25, T35, T45), using mobile phases containing acetonitrile or methanol, with and without ion-pairing reagents. Interestingly, the results reveal a clear correlation between Dm and molecular weight in the presence of ion-pairing, while no such trend is observed in the absence of ion pairing. It is also shown that Young's equation underestimates Dm values for ONs in non-ion-pairing conditions with a relative difference up to 150 %, while closely aligning with the experimental values in the presence of ion pairing reagents, suggesting the occurrence of conformational changes of ONs. This study provides a practical workflow for the accurate experimental determination of ON Dm values, which will be applied in future work to create a database of ON Dm data under varying chromatographic conditions to support the fundamental investigation of HPLC column performance for ON analysis.