Improved separation by at-column dilution in preparative hydrophilic interaction chromatography

Area of Science:

• Chromatography
• Analytical Chemistry
• Separation Science

Background:

• Preparative hydrophilic interaction chromatography (HILIC) often suffers from peak distortion due to strong solvent injection.
• At-column dilution (ACD) is explored as a technique to mitigate injection-related peak distortion.

Purpose of the Study:

• To evaluate the effectiveness of at-column dilution (ACD) in reducing peak distortion in preparative HILIC.
• To assess the impact of ACD on plate number and peak capacity under various elution and injection conditions.

Main Methods:

• Model compounds (uridine, cytidine, guanosine) were injected using conventional and ACD methods with varying aqueous-acetonitrile ratios.
• Plate number and peak capacity were measured under isocratic and gradient elution conditions.
• Separation of a plant root aqueous extract was performed to validate findings.

Main Results:

• In isocratic elution, ACD improved plate numbers for injections >40% water but decreased them for <40% water.
• ACD maintained efficiency with high-aqueous injections, unlike conventional methods.
• In gradient elution, ACD enhanced peak capacity with increasing water content and injection volume.

Conclusions:

• At-column dilution (ACD) is advantageous for preparative HILIC when injecting aqueous solutions.
• ACD offers improved loadability and peak shape preservation in both isocratic and gradient modes.
• This technique provides a valuable approach for handling challenging aqueous samples in preparative chromatography.