JOURACA_SP_2017

Effect of Mobile Phase Composition on
Steroid Retention in Reverse-Phase
Liquid Chromatography
Evan Li
Reversed-phase liquid chromatography is a
common technique used in analytical chemistry
for separating compounds in a solution.
In the chromatography experiment, a
mixture containing solutes to be separated is
dissolved in a liquid called the mobile
phase. The mobile phase is pumped
through a column containing a packed bed
of material, called the stationary phase. Differences
in interactions between solute, stationary
phase, and mobile phase cause different
solutes to exit the column at different
times. In particular, steroids such as cortisone,
hydrocortisone, prednisolone, and betamethasone,
whose structures are similar,
can be separated by liquid chromatography,
if separation conditions are optimized. Understanding
the influence of various chromatographic
variables on separation is key
to designing a successful chromatographic
method. In this experiment, we examined
the effect of three chromatographic variables
on the retention of the four previously
mentioned structurally-similar steroids:
mobile phase composition of a ternary solvent
system (water/methanol/acetonitrile),
temperature, and addition of a sugar molecule
called methyl-beta-cyclodextrin
(MBCD). We wish to observe how the steroid-cyclodextrin
complex is formed upon
addition of it to the mobile phase, and how
it affects retention of the steroids. Our hypothesis
is that through the addition of cyclodextrin,
these steroids will form a complex
that will lower its retention in the chromatographic
system. A change in the
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mobile phase composition and temperature
will also alter interactions between the steroids
and cyclodextrin, and consequently
will alter their retention rates. Cortisone
typically had the smallest retention factor
(abbreviated k') while betamethasone had
the largest. The general trend for all four
steroids was a decrease in k' by increasing
MBCD concentration and temperature. A
higher acetonitrile concentration significantly
lowered the k' among the steroids but addition
of MBCD and higher temperatures
did not seem to have a significant role as
compared to the solvent system with greater
methanol concentrations. Betamethasone
had the largest change in k' factor by altering
the MP.
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