Chromatography: The Principles Behind Every Variant

The single underlying idea

A mixture is carried by a mobile phase past a stationary phase. Each component distributes between the two according to its own equilibrium. A component that spends more time associated with the stationary phase travels more slowly, and given enough distance the components separate.

Everything else — the different techniques, the terminology, the equations — is refinement of that one sentence.

The main variants

The quantities that describe a separation

Retention factor

In planar chromatography, the retardation factor is the distance moved by the component divided by the distance moved by the solvent front. It lies between zero and one and is characteristic under fixed conditions.

In column chromatography the analogous quantity compares the time a component takes to elute with the time an unretained species takes.

Resolution

Two peaks are resolved when the distance between their centres is large compared with their widths. Both matter: peaks can be far apart yet unresolved if they are broad, which is why reducing band broadening is as important as increasing separation.

Plate theory

The column is modelled as a series of theoretical plates, each representing one equilibration. More plates means narrower peaks and better resolution. The plate height is the column length divided by the number of plates, so a smaller plate height indicates a better column — a point that is easy to state backwards.

The van Deemter equation

Plate height H depends on the mobile phase velocity u through three terms, each with a distinct physical origin:

• A — eddy diffusion. Different molecules take paths of different length through the packing. Independent of velocity, and reduced by uniform small particles.
• B/u — longitudinal diffusion. Molecules diffuse along the column while travelling. Worse at low velocity, because there is more time to diffuse.
• Cu — mass transfer resistance. Equilibration between phases is not instantaneous. Worse at high velocity, because there is less time to equilibrate.

Because one term falls with velocity and another rises, there is an optimum flow rate at which plate height is minimised. Being asked to explain why running a column faster does not always help — and why running it slower does not either — is answered entirely by this equation.

Choosing conditions

In normal phase chromatography the stationary phase is polar and the mobile phase relatively non-polar, so polar compounds are retained longer. Reversed phase inverts both, so non-polar compounds are retained longer. Reversed phase is far more common in practice because aqueous mobile phases suit most samples.

Knowing which mode is in use is essential before predicting elution order, and a question that specifies one is expecting the elution order to follow from it.

Frequently asked questions

Why do large molecules elute first in size exclusion?

Because they are excluded from the pores and travel only through the space between particles, which is a shorter path. Small molecules enter the pores and take longer.

What limits resolution in practice?

Band broadening. Two components may have quite different retention yet still overlap if their peaks are broad, which is why column efficiency matters as much as selectivity.

Why is there an optimum flow rate?

Because longitudinal diffusion worsens at low velocity while mass transfer resistance worsens at high velocity. The sum of the two terms has a minimum in between.

How does gas chromatography differ in principle?

Not at all in principle — only the mobile phase is a gas. The consequence is that the sample must be volatile and thermally stable, which limits what can be analysed.