different substances to migrate at different speeds. This is called partitioning, and it represents an
equilibrium between the two phases. Different compounds will have different partitioning
coefficients and will elute at different rates. This results in separation within the stationary phase,
allowing us to isolate each substance individually.
There are many different media that can be used as the stationary phase, each one exploiting
different properties that allow us to separate out the desired compound. On the MCAT, the property
most commonly used is polarity. For instance, thin-layer chromatography (TLC), which we will
shortly discuss, uses silica gel, a highly polar substance, as its stationary phase. Cellulose, another
polar substance, may also be used. This means that any polar compound will adhere to the gel well
and thus move through (elute) slowly. When using column chromatography, size and charge both
have a role in how quickly a compound moves through the stationary phase. Chromatography can
use even strong interactions, such as antibody–ligand binding.
As mentioned earlier, chromatography is based on the speed at which compounds move through
media. In practice, however, we will measure either how far each substance travels in a given
amount of time (such as in TLC) or how long it takes to elute (as in column or gas chromatography).
The types of chromatography that we will discuss include thin-layer and paper chromatography,
column chromatography, gas chromatography (also called gas–liquid chromatography), and high-
performance liquid chromatography, or HPLC.
THIN-LAYER AND PAPER CHROMATOGRAPHY
Thin-layer chromatography and paper chromatography are extremely similar techniques, varying
only in the medium used for the stationary phase. For thin-layer chromatography, a thin layer of
silica gel or alumina adherent to an inert carrier sheet is used. For paper chromatography, as the
name suggests, the medium used is paper, which is composed of cellulose.
For these techniques, the sample that we want to separate is placed directly onto the adsorbent
itself; this is called spotting because we apply a small, well-defined spot of the sample directly onto
the silica or paper plate. The plate is then developed, which involves placing the adsorbent upright
in a developing chamber, usually a beaker with a lid or a wide-mouthed jar. At the bottom of this jar
is a shallow pool of solvent, called the eluent. The spots of sample must be above the level of the
solvent, or else they will dissolve into the pool of solvent rather than running up the plate. When set