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By Rick Lake and Ty Kahler
• Column selectivity has the most significant influence
on chromatographic peak separation (i.e., resolution).
• Initially focusing on columns instead of mobile phases
will drastically speed up method development.
• Restek’s USLC™ column set boasts the widest range of
selectivity available—using just 4 stationary phases!
Equation 1: Selectivity is the driving parameter of resolution,
as it affects peak separation to the greatest degree.
Wasted effort. Lost time. Frustration. Making the wrong decisions can
needlessly complicate and delay successful method development. By
understanding selectivity’s impact on resolution and focusing on col-
umn choice to create alternate selectivity, you can drastically speed
up LC method development. Enter the new Restek Ultra Selective
Liquid Chromatography™ (USLC™) columns.
Change Your Habits—and Your Columns—to
Optimize Resolution
Resolution is the result of 3 cumulative terms: efficiency (N), retention
capacity (k), and selectivity (α). How well and how quickly we resolve
our analytes depends upon our ability to control these factors. Of
the 3, selectivity affects resolution to the greatest degree (Equation 1).
For that reason, any discussion about resolution in method develop-
ment should focus on selectivity.
All too often, HPLC method developers use C18 columns and rely on
adjusting mobile phases to alter selectivity and reach a desired sepa-
ration. While it is true that mobile phase adjustments may alter selec-
tivity, it is a laborious task that typically creates only marginal differ-
ences. In addition, some mobile phases are not practical with certain
detection modes, including mass spectrometry (MS) and refractive
index (RI). To save time and work, you should first focus on choosing
the right stationary phases (i.e., columns). Columns pose fewer issues
with MS and RI, change easily, and offer alternate and even orthogo-
nal separations for maximum effect with each change.
Choosing columns can be incredibly difficult, but by characterizing
stationary phase selectivity, we created new guidelines for easily
making the right choice.
The Highest Range of Alternate Selectivity
Using the hydrophobic subtraction model (H-S model) [1], we quanti-
fied the selectivity of our stationary phases and determined which
phases produce the greatest degree of dissimilarity compared to a
C18 benchmark. We then matched these phases with specific solute
types based on molecular interactions commonly encountered in
reversed phase chromatography. By doing so, we were able to (1) find
a small set of columns with the widest range of alternate selectivity
available and (2) recommend columns based on the chemical proper-
ties of target analytes.
Figure 1 illustrates the retention profile of a C18 compared with
those of the 4 Restek USLC™ columns. USLC™ phases are highly
selective and exhibit significantly different retention profiles based
on specific solute chemical properties, so you can match USLC™ col-
umns to specific analytes and accelerate method development!
To confirm the orthogonality of the Restek USLC™ column set, we also
quantified its selectivity (S) as described by Neue et al. [2] by looking
at the degree of scatter along a regression line when compared to a
conventional C18 (Figure 2). USLC™ phases produce the highest range
of alternate selectivity available today—using only 4 columns.
Summary
The Restek USLC™ column set has a profile that encompasses the
widest range of reversed phase selectivity available today. Instead
of manually altering mobile phases, operational parameters, or
instrument settings—often with minimal effect on resolution—take
advantage of the Restek USLC™ column set. These 4 orthogonal
stationary phases and their defined retention profiles let you quickly
determine the best column for almost any reversed phase situation.
Selectivity (S) = 100 x 1-r2
S = 53.5
R = ¼ N x ( k/(k+1)) x (α-1)
E ciency Retention Factor Selectivity
Simplify HPLC and UHPLC Method Development
With the Restek USLC™ Column Set