Characterization of reversed-phase liquid chromatographic columns containing positively charged functionality

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Existing RP column characterization methods fail to accurately assess low positive charges.
• Discriminating between neutral and low positive charge RP columns is challenging.

Purpose of the Study:

• To develop a robust chromatographic method for assessing low positive charges on RP columns.
• To introduce a new parameter (α4-OBSA/Tol) for column characterization.
• To aid in selecting equivalent or dissimilar RP columns for method development.

Main Methods:

• Utilized electrostatic attraction of 4-n-octylbenzene sulfonic acid (4-OBSA) and retention of toluene (Tol) to determine positive charge.
• Analyzed 15 commercial RP-LC columns.
• Combined new parameter with existing Tanaka parameters for column selection.

Main Results:

• Developed and validated the α4-OBSA/Tol parameter for quantifying low positive charge.
• Demonstrated that even neutral alkyl phases can exhibit positive charge at low buffer concentrations.
• Identified mobile phase conditions (low % MeCN) promoting mixed-mode retention.
• Categorized columns into three subsets based on positive charge character.

Conclusions:

• The new method and parameter (α4-OBSA/Tol) effectively characterize low positive charges on RP columns.
• The findings assist chromatographers in selecting appropriate columns for method development and biopharmaceutical analysis.
• The study highlights the importance of considering column charge characteristics in chromatography.