Quantifying injection solvent effects in reversed-phase liquid chromatography

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Injection solvent composition significantly impacts chromatographic peak shape and method sensitivity.
• Understanding the relationship between injection solvent and column properties is crucial for robust method development.

Purpose of the Study:

• To quantify the impact of injection solvent strength, volume, and mass on peak distortion in liquid chromatography.
• To define and measure method sensitivity to injection solvent strength.
• To correlate column parameters with observed injection solvent sensitivity.

Main Methods:

• Peak distortion was measured across various injection parameters (solvent strength, volume, mass, retention factor, column selectivity).
• Method sensitivity (s) was mathematically defined using theoretical plate counts.
• Column parameters were determined using the hydrophobic-subtraction model and acetonitrile excess adsorption isotherm.

Main Results:

• Near-ideal sensitivity (s>0.90) was achieved with low-volume (1.25 μL) injections across all columns and analytes.
• Increased injection volume had a greater negative impact on sensitivity than increased injection mass.
• Column properties like decreased ligand density and increased silanol activity correlated with consistent peak shapes.

Conclusions:

• Low-volume injections are critical for maintaining high method sensitivity in liquid chromatography.
• The ratio H/A from hydrophobic-subtraction parameters can predict a column's sensitivity to injection solvent.
• The study provides a framework for predicting and mitigating injection solvent effects for method optimization.