The effect of column and eluent fluorination on the retention and separation of non-fluorinated amino acids and proteins by HPLC

The effect of column and eluent fluorination on the retention and separation of non-fluorinated amino acids and proteins in HPLC is investigated. A side-by-side comparison of fluorocarbon column and eluents (F-column and F-eluents) with their hydrocarbon counterparts (H-column and H-eluents) in the separation of a group of 33 analytes, including 30 amino acids and 3 proteins, is conducted. The H-column and the F-column contain the n-C8H17 group and n-C8F17 group, respectively, in their stationary phases. The H-eluents include ethanol (EtOH) and isopropanol (ISP) while the F-eluents include trifluoroethanol (TFE) and hexafluorosopropanol (HFIP). The 2 columns and 4 eluents generated 8 (column, eluent) pairs that produce 264 retention time data points for the 33 analytes. A statistical analysis of the retention time data reveals that although the H-column is better than the F-column in analyte separation and H-eluents are better than F-eluents in analyte retention, the more critical factor is the proper pairing of column with eluent. Among the conditions explored in this project, optimal retention and separation is achieved when the fluorocarbon column is paired with ethanol, even though TFE is the most polar one among the 4 eluents. This result shows fluorocarbon columns have much potential in chromatographic analysis and separation of non-fluorinated amino acids and proteins.

Graphical abstractFluorocarbon column and fluorocarbon eluents are compared side-by-side with their hydrocarbon counterparts to extract the effect of column and eluent fluorination on analyte retention and separation. Statistical analyses of retention time data revealed that optimal retention and separation is achieved when the fluorocarbon column is paired with ethanol.Figure optionsDownload full-size imageDownload as PowerPoint slideResearch highlights▶ Fluorocarbon column and eluents are compared with their hydrocarbon counterparts. ▶ Separation of non-fluorinated amino acids and proteins were analyzed. ▶ Proper pairing of columns and eluents is critical for best retention and separation. ▶ Optimal result is achieved when a fluorocarbon column is paired with ethanol.