For the last several months we've been working through the steps to develop a liquid chromatography (LC) method quickly and
effectively. First we looked at setting separation goals (1) and selecting the starting conditions (2). Then we adjusted retention
times (3) and mobile phase conditions (4) to get the retention and peak spacing to meet the goals we set. Changes in the mobile
phase percent organic (%B), solvent type, pH, and temperature were easy variables to modify in an effort to fine-tune the
separation, because these variables can be changed in a continuous manner. That is, the incremental change in the parameter
from one run to the next can be made in any step size we desire, such as a change from 45% B to 46% B or 43% methanol, 35%
acetonitrile, and 22% buffer to 44% methanol, 34% acetonitrile, and 22% buffer. One other variable that can be useful to change
peak spacing is a change in the column type, for example, C18 to phenyl. Unfortunately, such column changes are in discrete
steps — it is not possible to change from 44% phenyl and 56% C18 to 43% phenyl and 57% C18. And changing columns is expensive
— typically $500 per column — so column changes have more budgetary impact than changes in pH or temperature.
This month's installment of "LC Troubleshooting" will focus on changing the column as a means to change the peak spacing in
a chromatogram. We will consider two approaches — a traditional one of selecting the column by bonded phase type and a newer
technique based upon the chromatographic properties of the column.
"Orthogonal" Columns
John W. Dolan
We often hear the term "orthogonal" to describe a column or separation change in the quest to obtain a better separation of
two or more peaks. Strictly speaking, orthogonal conditions are those that produce a separation that is at right angles or
perpendicular to the current one. As long as we are working with reversed-phase LC, hydrophobic interactions dominate the
separation mechanism, so no matter what change we make, hydrophobic interactions are still the most important ones. As a result,
there is no truly orthogonal separation condition in this context. Perhaps if we switched to a different retention mechanism,
such as from reversed phase to ion exchange, we might get orthogonality, but some would argue that as long as we used LC as
the analytical tool, we wouldn't achieve orthogonal results.
Our present goal is to get a significantly different separation than the one we currently have, and in this context, we'll
refer to a set of conditions that achieves this goal as orthogonal. (Those of you who are purists had better stop reading
at this point or take your blood pressure medicine!)
Contributions to Column Selectivity
There are three major contributions to achieving the desired selectivity, or peak spacing, in reversed-phase LC, the analyte
chemistry, the mobile phase composition, and the column composition. For the most part, we're stuck with the analyte chemistry
(with the major exception for ionic compounds when the mobile phase pH is changed), and we've already explored mobile phase
changes. The column chemistry has two major contributions — the packing particles (usually silica) and the bonded phase. There
was a time when we thought all silica was created equal and all bonded phases of the same description were the same. Thus,
a C18 column was a C18 column . . . period. This gave rise to the L-1 classification in the United States Pharmacopoeia (USP), grouping all C18 columns in one category. Now, unless you are very new at LC or very naive, you realize that not all
C18 columns are created equal.
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