Overloading study of basic compounds with a positively charged C18 column in liquid chromatography.

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Reversed-phase (RP) chromatography often faces challenges with basic compound separation due to tailing and overloading.
• Positively charged stationary phases offer a potential solution for improved retention and peak shape of basic analytes.

Purpose of the Study:

• To evaluate the surface charge properties of the novel XCharge C18 column.
• To investigate the overloading behavior of basic compounds on this positively charged RP column.
• To determine the influence of mobile phase conditions on column performance and loadability.

Main Methods:

• Surface charge evaluation using nitrate ion retention across varying pH and buffer concentrations.
• Systematic study of overloading using amitriptyline as a basic probe.
• Analysis of peak shape, loadability (C(0.5)), and column efficiency under different mobile phase conditions.

Main Results:

• The XCharge C18 column exhibits a significant, pH-dependent positive surface charge.
• Excellent peak shapes (Tf<1.5) and high loadability (C(0.5) ≈ 30,000 mg/L) were achieved for bases with 0.1% formic acid.
• Phosphate buffers and higher pH reduced loadability and worsened peak shape at higher sample loads, despite potential efficiency gains at low loads.

Conclusions:

• The XCharge C18 column demonstrates superior performance for basic compound separation, particularly alkaloids.
• Appropriate ionic repulsion is crucial for optimal performance, explained by the multiple-site adsorption theory.
• Careful selection of mobile phase pH and buffer composition is essential for maximizing loadability and peak shape on this column.