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.
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