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It is the ratio of capacity factors for two chromatographic peaks.
Conceptually, a capacity factor is the ratio of the amount of time
an analyte spends in the stationary phase to the amount of time
it spends in the mobile phase. Since all analytes spend the same
amount of time in the mobile phase (equal to the dead time t0),
selectivity is the ratio of the amount of time the later eluting ana-
lyte spends in the stationary phase relative to that of the earlier
eluting analyte. While the mobile phase composition in liquid
chromatography can be varied to encourage an overall greater
or lesser retention, the primary factor controlling selectivity is
the ability of the stationary phase to differentially interact with
each analyte. The primary means to alter selectivity in a chro-
matographic separation is to change the stationary phase or the
mode by which analytes interact with the stationary phase.
While different separation modes (e.g., reversed phase [RP],
hydrophilic interaction [HILIC], aqueous normal phase [ANP],
normal phase [NP], etc.) can be used to affect the ways that ana-
lytes interact with a given stationary phase, we confine ourselves
here to discussions on RP separations. Virtually every chemistry
student has experience in RP separations—most likely focused
on generic separations using an octadecylsilyl (C18-bonded
silica gel) bonded phase. The first thing to note is that all C18
phases are not created equal. Changes in the underlying sup-
port chemistry, the way bonded groups are attached to the
support, and the ways potentially deleterious interactions with
residual silanol groups are shielded, significantly affect the reten-
tion of different analytes. For example, amine-containing com-
pounds often exhibit significant tailing in chromatograms if they
can interact with silanol groups. The strategy is to induce a uni-
form dominant interaction mode between the analyte and the
stationary phase so that nicely symmetrical peaks are observed.
For a typical C18 phase, the dominant interaction is induced by
the hydrophobic effect. Significant differences in the hydropho-
bic content in chemical structures allow the C18 phase to exert
selective interactions with each analyte and, assuming adequate
retention and good efficiency are maintained, chromatographic
resolution will result.
Complex mixtures will contain a multitude of chemical com-
pounds that possess variable physicochemical properties.
Oftentimes, the chromatographer is concerned with the qualita-
tive and quantitative speciation of multiple analytes from a sin-
gle class (e.g., polyphenols, drugs and their metabolites, steroids,
etc.). If each compound has a different molecular weight, one
might be able to bypass the need for chromatographic resolu-
tion of all components of interest by using a selective detector,
such as a mass spectrometer. However, a mass spectrometer
cannot directly differentiate compounds that have the same
mass, and many analytes in a class of compounds may simply
be isomers, which have the same elemental formula. While it
is possible to use some tandem mass spectrometry approaches
to differentiate coeluting isobaric compounds, the most reliable
means by which to differentiate them for speciation would be to
chromatographically resolve them prior to detection. A generic
C18 phase may not provide sufficient selectivity to accomplish
this task.
Those who move beyond college course-based laboratory exer-
cises will quickly learn that there are other stationary phases
available to impart additional selectivity in reversed-phase sepa-
rations. Recent moves to alter support chemistries, including the
use of superficially porous particles, have a major impact on effi-
ciency of separations. However, to impact changes in selectivity,
more important are changes in the chemistry of moieties bonded
to these supports. Different manufacturers offer a milieu of
alternatives that can range from the incorporation of polar units
imbedded in the C18 chain or the bonding of different functional
units all together. A favorite question I ask my senior-level instru-
mental analysis class is,“How can a cyano-bonded phase be used
in both NP and RP separation modes?” The cyano phase is ideal
for NP separations where a polar stationary phase is paired with
a nonpolar mobile phase. However, in reversed-phase mode, this
polar phase can impart vastly different retention interactions to
more polar analytes compared to a C18 phase. This can cause
large changes in elution order for a mixture of analytes because
the cyano group provides a vastly different selectivity, and it
is still effective for use in RP mode with a polar mobile phase.
Similarly, use of phases that incorporate polar groups embed-
ded somewhere along a C18 chain enable hydrogen-bonding
interactions to assist in selective retention of different compound
classes. Care should still be taken that these interactions are uni-
form and do not impart poor peak shape due to non-uniformity
of chromatographic separations (similar to silanol effects), but
for certain classes these additional interaction sites can be the
difference between separation or coelution. Available now are
also biphenyl phases which, in the presence of the right mobile
phase, exert pi-interactions that can improve selectivity and
retention for aromatic analytes. Interestingly, a biphenyl phase
will exert these interactions in the presence of an aqueous meth-
anolic mobile phase, but in the presence of acetonitrile, which
itself has a strong pi-character, the phase will behave more like a
C18. The change in selectivity can be quite dramatic.
The chromatographer’s toolbox is ever expanding. Sometimes
this can be overwhelming. Manufacturers have given different
generic (and sometimes difficult to interpret) names to the dif-
ferent stationary phase supports and bonded phases they use
to create their products. Luckily, they also spend a great deal
of time and effort providing educational materials to guide the
choice of the proper phase for different applications. Even so,
one should always go back to the master resolution equation to
reason the underlying fundamentals that will eventually yield
separation of target compounds of interest. Chemists and bio-
chemists will never stop creating new chemical compounds, and
we are still figuring out the chemical diversity provided by nature.
Thus, analytical chemists will always have a job in characterizing
new analytes or determining their presence in various systems. It
is a good thing that there are a lot of choices in the tools that one
can use to accomplish these tasks.