pH gradient reversed-phase HPLC

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Conventional gradient HPLC relies on organic modifier concentration changes.
• Ionogenic analytes present unique separation challenges in reversed-phase HPLC.
• Existing methods may be unsuitable for analytes sensitive to high organic solvent concentrations.

Purpose of the Study:

• Introduce and describe pH gradient HPLC as a new separation mode.
• Develop a theoretical framework and mathematical model for pH gradient HPLC.
• Demonstrate the practical applicability and advantages of pH gradient HPLC.

Main Methods:

• Implemented pH gradient elution by linearly changing eluent pH during the run.
• Maintained a fixed organic modifier content in the mobile phase.
• Developed and validated a mathematical model to predict retention and separation.

Main Results:

• pH gradient HPLC effectively separates ionogenic analytes by altering analyte dissociation.
• Achieved reduced peak width and minimized peak-tailing, particularly for organic bases.
• The theoretical model accurately predicted experimental outcomes in gradient and isocratic conditions.
• Demonstrated utility for analytes sensitive to organic solvents, including bioanalytes.

Conclusions:

• pH gradient HPLC is a versatile and effective technique for ionogenic analyte separation.
• The developed mathematical model provides a rational basis for optimizing separations.
• This method offers advantages for sensitive analytes and aids in determining physicochemical parameters like pK(a).