The Hydrophobic-Subtraction Model and Reversed-Phase
Selectivity - A Simplified Look at Column Phase Selection
Ty Kahler, Rick Lake, Steve Kozel, Mike Wittrig, Bruce Albright, Chris Denicola
Restek Corporation, Bellefonte, PA, USA
Abstract
Results
Previous work has suggested that the selection of “orthogonal” columns can be predicted using the values
determined from the hydrophobic-subtraction model [1]. This work is of importance as the demands of
chromatographic separations, like sample throughput, number of anayltes, and sample matrix, becomes more
stringent. Column selectivity has a significant influence on chromatographic peak separation, or resolution, so
choosing the proper column can greatly enhance HPLC and UHPLC method development. This presentation will
demonstrate a guideline for reversed-phase (RPLC) column phases and phase-solute interactions to aid in choosing
the most selective columns for methods development.
Once an orthogonal column set was defined, and the contributions of silica were kept constant, we could then
characterize the types and extent of intermolecular interactions as they contribute to retention differences in stationary
phases, or selectivity. Table 1 illustrates the characterization of phase-solute interactions largely contributing to
reverse phase selectivity in our defined column set and the calculations employed to quantify the results of the
hydrophobic-subtraction model.
Phase-Solute
Interaction
Solute Retention
Characteristic
Description
Value
Scope
Dispersion
Hydrophobic
Van der Waals interactions that exists to some extent in all
organic molecules.
H Term of Hydrophobic Subtraction model
Charge Transfer /
π- Acidity
Aromatic
•Solutes deficient in electrons interact with Lewis base phases
k Anisole / k Ethyl benzene
Hydrogen
Bonding
Acidic
•Solute acts as proton donor with proton accepting
functionality embedded in the stationary phase.
B Term of Hydrophobic Subtraction model
•Solutes rich in electrons interact with Lewis acid phases or
phases with an induced dipole.
C Term of Hydrophobic Subtraction model
Using the testing protocol of the hydrophobic-subtraction model we will empirically define column classes as they
pertain to retention profiles. By extending this treatment to define solute retention for substituted alkyl and non-alkyl
phases, we will define an orthogonal column set to exploit column selectivity in reversed phase columns. Finally, this
extended treatment will provide a simplified guideline for column selection and create a needed link between analyte
chemistry and stationary phase.
Table 1 Phase-solute Interactions contributing to Reversed Phase Selectivity
Induced Dipole or Basic
π-Basicity
Experimental Design
The conditions and calculations described in the hydrophobic-subtraction model were followed to define a basis for
stationary phase and silica contributions to selectivity, as well as a qualitative approach to column equivalency.
Figure 1 illustrates the test probes and calculations used to define silica and stationary phase contributions to
retention.
Figure 1 Parameters and Probes of the Hydrophobic-Subtraction Model
The retention profiles of our experimental column set were then quantified using the above values to compare the
phase-solute contributions as they relate to a monomeric C18. We could then draw a correlation to the preferential
solute retention and analyte type. Ultimately, we can define a guideline for practical column selection based on
analyte type. Figure 4 Illustrates the retention comparison of our defined column set by analyte type.
Figure 4 Retention Profiles of Experimental Column Set Relative to Analyte Type
Hydrophobic Retention
Aromatic Retention
Acid Retention
Base Retention
List of Terms :
Hydrophobicity (H)
hydrophobic retention
Steric Resistance (S*)
resistance to penetration of molecules into
stationary phase
Column hydrogen-bond acidity (A)
free silanol/siloxanes
(Proton donating)
Column hydrogen-bond basicity (B)
free silanol/siloxanes
(Proton accepting)
Column cation-exchange activity (C)
ionized silanols – charge measure
(pH dependant)
From the Fs value, we empirically determined the column “dissimilarity” as the reverse of column equivalency,
relative to a monomeric C18. For these experiments, the base silica (Ultra column line) was kept constant to not add
dissimilarity from changes in the silica, and as a means to focus on the contributions of stationary phase on the A,B
and C terms. This, then defined the practical column set for further experimentation. Figure 2 illustrates the equation
for column equivalency and the experimental results used to define column dissimilarity. Figure 3 shows the
experimental column set used to further define phase-solute interactions
Figure 2 Quantifying Column Dissimilarity
We can then further quantify the range of selectivity as described by Neue et al. [2]. By looking at the retention
characteristics of the H-S model solute probes, we can define selectivity as the degree of scatter along the regression
line when comparing stationary phases to the conventional C18 benchmark. Figure 5 Illustrates a regression plot of
selectivity and the equations used to quantify the selectivity range of our orthogonal column set.
Figure 5 Regression Plot of Solute Probe Retention of Defined Column Set to Quantify Selectivity
Selectivity Range of Column Set
Figure 3
Experimental Column Set
Stationary
Phase Type
and Column
Class
Conclusion
The hydrophobic-subtraction model can be extended to describe the phase-solute interactions of modern reversed
phase columns. This can prove to be a useful tool in defining a column set to provide the widest possible range of
selectivity within the smallest column set. This could offer a simplified approach to practicing method developers. We
were able to demonstrate how knowledge of reversed-phase (RPLC) column phases and phase-solute interactions
can aid in choosing the most selective columns for column screening, solute confirmation, and methods development.
[1] L.R. Snyder, J.W. Dolan, P.W. Carr, The Hydrophobic-Subtraction Model of Reversed-Phase Column Selectivity, J. Chromatogr. A 1060 (2004) 77.
[2]
U.D. Neue, J.E. O’Gara, A. Mendez, Selectivity in Reversed-Phase Separations Influence of the Stationary Phase, J. Chromatogr. A 1127 (2006) 161.