Retention time and peak width in the combined pH/organic modifier gradient high performance liquid chromatography

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Reversed-phase high-performance liquid chromatography (RP HPLC) is crucial for separating complex mixtures.
• Predicting chromatographic behavior under gradient conditions, especially with combined pH and organic modifier changes, remains challenging.

Purpose of the Study:

• To evaluate the applicability of existing theory for predicting peak retention time and width in RP HPLC using combined pH/organic modifier gradients.
• To develop and validate a predictive model for RP HPLC separations.

Main Methods:

• Conducted 38 isocratic measurements across a wide pH and methanol range for ketoprofen and papaverine.
• Developed a model to describe retention factor and height equivalent of a theoretical plate (HETP) dependence on pH and organic modifier content.
• Simulated and compared retention times and peak widths for various isocratic and gradient conditions (methanol, pH, combined).

Main Results:

• The developed model accurately described experimental data for retention factor and HETP.
• Simulations based on isocratic data closely matched experimental retention times and peak widths for various gradient types.
• A simplified model, parameterized with 12 initial experiments, also showed excellent agreement with experimental data.

Conclusions:

• The proposed modeling approach effectively predicts analyte retention times and peak widths under varying pH and organic modifier conditions in RP HPLC.
• The simplified model offers a promising, efficient method for optimizing RP HPLC separations, particularly for complex samples and peak compression strategies.