Multiple, simultaneous, independent gradients for versatile multidimensional liquid chromatography. Part I: Theory

Area of Science:

• Analytical Chemistry
• Chromatography
• Separation Science

Background:

• Traditional liquid chromatography (LC) often uses coupled gradients, limiting independent control over separation parameters.
• Dual simultaneous dependent gradients (DSDGs) in LC alter multiple mobile phase conditions simultaneously but lack independent variability.
• This limitation hinders optimal chromatographic resolution for complex analyte mixtures.

Purpose of the Study:

• To develop a theoretical framework for uncoupling multiple gradients in LC.
• To enable independent control of dual simultaneous gradients for enhanced chromatographic performance.
• To predict the impact of independent gradients on the separation of complex mixtures, particularly proteins.

Main Methods:

• Developed a theory for uncoupling n gradients (n ≥ 2) in LC.
• Proposed using 2(n) reservoirs and a specialized eluent delivery system.
• Derived equations for freely apportioning flows among reservoirs to achieve independent gradients.

Main Results:

• Demonstrated the theoretical possibility of uncoupling dual simultaneous gradients.
• Predicted substantial increases in chromatographic resolution using dual simultaneous independent gradients (DSIGs) of salt and pH.
• Explained the prediction using electrostatic interaction theory for protein binding.

Conclusions:

• Uncoupling dual gradients in LC is theoretically achievable and essential for optimal separation.
• Dual simultaneous independent gradients (DSIGs) offer a significant advantage in resolving complex mixtures.
• The developed theory provides a foundation for advanced LC methods, particularly for protein separation.