Automated Programmable Generation of Broad pH Range Volatile Ionic Eluents for Liquid Chromatography

Area of Science:

• Analytical Chemistry
• Chromatography
• Biochemistry

Background:

• Universal detectors in liquid chromatography (LC), such as mass spectrometry, require complete eluent volatilization.
• Biomolecule separation necessitates aqueous mobile phases with controlled pH and ionic strength to preserve biological activity.
• Conventional eluents like ammonia/acetic/formic acids have limitations in concentration, pH buffering range, and nonvolatile components.

Purpose of the Study:

• To develop an automated eluent generation system for LC using gaseous precursors.
• To overcome limitations of conventional eluents for biomolecule separation.
• To explore applications in separating amino acids, proteins, monoclonal antibodies, and chiral amines.

Main Methods:

• Automated pressure-programmed generation of eluents from high-purity gaseous ammonia (NH3) and carbon dioxide (CO2) via gas-permeable membranes.
• Utilizing formic/acetic acids in the mobile phase to extend the low pH limit.
• Investigating fundamental pH, ionic strength, and buffer intensity considerations for the generated eluents.

Main Results:

• Successful application of the novel eluent system for the separation of amino acids, proteins, and monoclonal antibodies.
• Demonstrated use of dissolved CO2 as an effective ion-pairing agent for chiral amine separation.
• The system allows for precise control over eluent properties, overcoming limitations of traditional methods.

Conclusions:

• The developed automated eluent generation system offers a versatile and effective approach for LC separations, particularly for biomolecules.
• This method addresses the need for volatile, controllable eluents in aqueous media, preserving analyte integrity.
• The system expands the capabilities of LC for complex separations, including chiral analysis.