Retention model for proteins in reversed-phase liquid chromatography

Area of Science:

• Analytical Chemistry
• Chromatography
• Biochemistry

Background:

• Reversed-phase chromatography (RPC) is crucial for protein separation.
• Understanding protein retention mechanisms is essential for optimizing separation.
• Existing models may not fully capture the complexity of protein-surface interactions.

Purpose of the Study:

• To develop and validate a novel retention model for proteins in RPC.
• To establish a quantitative relationship between protein properties and chromatographic retention.
• To investigate the influence of mobile phase composition on protein retention.

Main Methods:

• Development of a retention model based on solvent displacement (Z value).
• Experimental validation using various proteins and mobile phase compositions (e.g., formic acid, alcohols).
• Analysis of protein capacity factor, molecular weight, and desorption curves.

Main Results:

• The retention model accurately predicts protein retention based on the number of solvent molecules (Z) displacing the solute.
• A direct proportionality was observed between Z and protein molecular weight using 60% formic acid.
• Protein desorption curves showed increased convexity with higher molecular weights, aligning with the model's predictions.
• The Z number varied with solvent type (alcohols) and mobile phase additive concentration.

Conclusions:

• The proposed retention model provides a robust framework for understanding protein behavior in RPC.
• Protein retention is strongly correlated with molecular weight and surface area interactions.
• The model's predictive power is influenced by mobile phase composition, offering insights for method development.