TITLE:
Optimization of High-Performance Liquid Chromatography Parameters for Purification of Oligonucleotide-A
AUTHORS:
Christina Perez, Mubeen Rani, Tuan Phan
KEYWORDS:
Purification, High-Performance Liquid Chromatography, Oligonucleotide-A, Dipotassium Phosphate, Triethylammonium Acetate, Triethylammonium Bicarbonate, Ammonium Acetate
JOURNAL NAME:
American Journal of Analytical Chemistry,
Vol.13 No.2,
February
28,
2022
ABSTRACT: The modification of high-performance liquid chromatography parameters leads to a more effective oligonucleotide-A purification process. Using various experimental parameters such as buffer, concentration, and pH, a method for optimizing the purification of an oligonucleotide-A on a reverse-phase C18 column was created. To purify oligonucleotide-A, High-Performance Liquid Chromatography (HPLC), Ultraviolet-Visible Spectrophotometry (UV-Vis), Liquid Chromatography-Mass Spectrometry (LC-MS), and lyophilization were used. Chromatographic data were collected with a semi-prep HPLC system, quantified with the UV-Vis technique, and validated with the LC-MS method. The most optimized parameters found to obtain the purity of 93.0% are 40 mM triethylammonium bicarbonate (TEAB) buffer with pH 7, which is approximately 6.0% higher than the reported method of which the purity is 87.0%. However, the yield under these conditions was reduced by about 5%. The worst possible optimized settings that resulted in the lowest purity (84.0%) and yield (69.0%) are 10 mM ammonium acetate (NH4CH3CO2) with pH 7.