Study of overload for basic compounds in reversed-phase high performance liquid chromatography as a function of...

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Understanding the overloading properties of stationary phases is crucial for optimizing chromatographic separations.
• Hybrid silica-octadecylsilica (ODS) phases offer unique selectivity but their loading behavior requires detailed investigation.

Purpose of the Study:

• To investigate the retention and overloading characteristics of a hybrid silica-ODS phase (XTerra RP-18) across a wide pH range (2.7-10.0).
• To evaluate the impact of pH and solute ionization on the loading capacity and efficiency of the chromatographic column.

Main Methods:

• Utilized eight basic solutes and two quaternary ammonium compounds.
• Employed phosphate and carbonate buffers to control pH from 2.7 to 10.0.
• Analyzed retention and efficiency as a function of sample mass and pH.

Main Results:

• At low pH, the hybrid phase exhibited overloading similar to pure silica and polymeric phases, with efficiency loss above ~0.5 microg.
• Loading capacity significantly improved at pH 7-8.5, attributed to reduced solute ionization.
• Loading capacity generally increased up to pH 10 for most bases, correlating with decreased ionization.
• High pH negatively affected peak shape for solutes with high pKa, potentially due to increased silanol ionization.

Conclusions:

• The hybrid silica-ODS phase shows pH-dependent loading capacity, with improved performance at neutral to moderately alkaline conditions.
• Solute ionization state is a key factor governing overloading behavior on this phase.
• Potential limitations exist at high pH for certain basic analytes due to silanol group activity.