This is the second instalment in a series on how to develop liquid chromatography (LC) methods in an efficient manner. Last
month,1 we considered how to set goals for new methods. This month, we will look at some of the factors involved in selecting a
starting point for method development. Although our focus in this series is on method development, in the spirit of LC troubleshooting,
we need to remember that many of the choices we make during method development will determine some of the problems that may
be encountered or avoided with the final method. So each choice of a specific parameter to optimize should be made with a
consideration of what kind of problems might occur during method development and with the completed method.
Playing the Odds
The first choice that we have to make in method development is which chromatographic mode we will use. There are reversed-phase,
normal-phase, hydro-philic interaction chromatography (HILIC), ion-exchange, size-exclusion, chiral and other modes from which
we can choose. For most of us in the pharmaceutical, environmental and chemical industries, the choice will be reversed-phase
LC. I look to Ron Majors' "Column Watch" reviews of the Pittsburgh Conference each spring as a finger in the wind in terms
of favoured column technology. Year after year, you'll see that the most common columns, either in terms of overall use or
new product introductions, are reversed-phase columns. The reasons are simple — they provide the necessary separation power
for a majority of separation problems, are easy to use and are reasonably robust. If I were a gambling man, I'd lay my money
down on the reversed-phase bet every time, unless I had a solid reason to choose otherwise.
Some obvious applications require other chromatographic modes. If your sample contains chiral compounds, you need a chiral
column, chiral mobile phase, or chiral derivative to enable the separation — reversed-phase LC just won't work. If you need
to maintain biological activity of an enzyme or other biomolecule, you will avoid reversed-phase LC because of its strongly
denaturing mobile phases. Separation of ionic compounds, particularly inorganic ions, will generally go better with ion-exchange
or ion chromatography. The separation of positional isomers is difficult by reversed-phase LC, but generally straightforward
by normal phase. So if your samples have special characteristics that preclude use of reversed-phase techniques, use common
sense and go with the chromatographic mode that is most likely to lead to success. But for the vast majority of compounds,
reversed phase is the best place to start. Continuous or Discontinuous?
Table 1: Ranking the variables.
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Now that we've decided upon reversed phase as our starting column type, we need to think a bit about the strategy we will
use to get a reversed-phase method. There are several variables that we can use during the development process. We need to
choose wisely to make the most out of our investment of time and money. One way to classify the parameters is whether they
are continuously variable or not, as listed in Table 1. Continuous variables are those that can be changed in infinitely small
steps, which gives an advantage in fine-tuning the separation and generally makes them more convenient to use. As the concentration
or magnitude of a continuous variable is changed, retention changes in a regular fashion, generally in a linear or logarithmic
manner. Discontinuous variables are those that can be changed only in a stepwise fashion, and as a result, retention does
not change in a continuous manner. Let's consider the list in Table 1.
Solvent strength: By solvent strength, we mean the amount of the strong solvent in the mobile phase, usually methanol, acetonitrile, or tetrahydrofuran
in reversed-phase LC. This is also referred to as percent B-solvent (%B). Of course, we can vary the %B in any increment we
want.
Temperature: Temperature can be varied most easily from a few degrees above room temperature to the limit of the column or column oven.
This means temperatures in the 30–70 °C range for most systems.
