Molnar-Institute: Theory of RPC Modeling

Theory of RPC Modeling

Retention phenomena of reversed-phase chromatography (RPC) are described in many ways by different authors. One of the groups attempting to understand the fundamentals of RPC were the developers of DryLab® software, LC Resources Inc. in United States, under the leadership of Lloyd Snyder. Other members of this group were John Dolan and Tom Jupille (Walnut Creek, California) and Imre Molnár (Berlin, Germany).
 
DryLab draws on the philosophy described in the "Solvophobic Theory" of Csaba Horváth, which was developed in the years 1975-1977 at Yale University (see Research/Publications of Dr. Imre Molnar).   

The fundamental concept of this theory is that retention in RPC is enforced by water, as a component of the eluent. The dissolution of nonpolar molecules in water requires large amounts of energy. The retention factor k (also called the "capacity factor") is proportional to the energy needed in the dissolution process. In the case of dibenzanthracene on a C8-phase, for example, is

in water, k ≈ 4000
in acetonitrile k ≈1
(see DryLab example files, PNAH.dlb)

Horváth and his team found that the only possible explanation for this extremely wide scale of retention is the change in the surface tension from water to acetonitrile (AN) or methanol (MeOH).   

Water is strongly lipophobic. However the lipophobicity of water can be easily and continuously reduced by admixing MeOH or AN to it.  

That is what we do in gradient elution.  

Gradient elution starts most frequently with water or with water-rich eluents. Upon injecting the sample into such a mobile phase (eluent), the water forces a reduction of cavities around the more or less hydrophobic sample components and brings them onto the surface of the C8 or C18 column packings. By increasing the amount of the organic component (MeOH or AN), the retention force from water will become weaker, the surface tension of the eluent is reduced from approximately 72 dyn/cm in water to approximately 20 dyn/cm in MeOH or AN, and the retention time is reduced at the same time. The process has tremendous capabilities for separating complex mixtures in a highly reproducible manner.

For gradient elution, DryLab calculates the retention precisely for every component. Based on only two gradient runs, DryLab can show, by switching to the isocratic mode, how the k-values are reduced with increasing %B (percent organic) in the mobile phase.

The amazing ease of reversed-phase gradient elution is exhibited in the continuous reduction of the retention force of water by increasing the amount of the organic eluent component (MeOH or AN) in gradient elution. Fine differences in accessible solvophobic molecular surface areas, combined with steps in the gradient, are sufficient to achieve reasonably good separations with almost any mixture in life science applications.

Modeling of reversed-phase separations by DryLab is based on the measurement of both the retention time and the capacity factor. The consequent calculation of sample positions in the corresponding chromatograms enables the chromatographer to look at experiments in a virtual mode and generate an overview of separation choices in minutes. If measurements are precise and reproducible, the retention behavior in complex mixtures can be modeled with high precision in seconds, saving valuable time in the expensive laboratory environment.

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