Document Type
STEM
Date
2023
Submission Date
August 2023
Abstract
This study focused on the application of reverse-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC is used for the purification of organic molecules, including pharmaceuticals, natural products, and proteins. We developed a modified version of a United States Pharmacopeia (USP) procedure to separate and purify the chemical components found in Excedrin tablets. The goal was to investigate the relationship between retention time (Tr) and the capacity factor (k') of solutes. By varying the composition of the mobile phase and stationary phase, we experimentally determined the capacity factor of acetaminophen (APAP), caffeine, and aspirin. Our results show that the capacity factor (k') of all solutes increased as the percentage of water in the mobile phase increased. This trend was consistent across all three columns tested. We also observed co-elution of solutes in the C8 and phenyl columns at lower water compositions. For superior separation and baseline resolution of all peaks, we found that the C18 column, as specified in the USP monograph, provided excellent results at all mobile phase compositions tested. Additionally, we investigated the solute retention as a function of mobile phase acidity. When using an acidified mobile phase with the C18 column (3% acetic acid), all solutes exhibited excellent peak shape and symmetry. However, the removal of acid from the mobile phase resulted in poor peak symmetry (non-Gaussian) for solutes containing a carboxylic acid moiety, such as aspirin and benzoic acid. Conversely, more polar solutes like APAP and caffeine maintained their Gaussian peak shape.In conclusion, our study highlights the successful implementation of RP-HPLC for the purification of organic molecules and reveals the relationship between retention time and the capacity factor of solutes. The choice of column and mobile phase composition played a crucial role in achieving optimal separation and peak symmetry.
Rights
© The Author(s)
Is Part Of
Auctus
DOI
https://doi.org/10.25886/bhe9-4z37