Solutions for Liquid Chromatography Exercises

1. Reversed-phase HPLC means that the stationary phase is less polar than the
mobile phase.
2. A guard column is a short column packed with the same material as the main
column and placed between the injection port and the main column. It serves to
remove impurities that might bind irreversibly to the main column and degrade its
efficiency.
3. A gradient elution refers to a liquid chromatography separation in which the
mobile phase composition changes during the elution. The mobile phase
composition is changed so that the mobile phase becomes more like the
stationary phase during the elution.
4. Note that the detector signal is the absorbance at 254 nm, and that A = lc. The
detector pathlength is constant, so the absorbance for toluene is smaller for a
larger concentration because the molar absorptivity () for toluene is much less
than that for acetophenone at this wavelength.
5. All samples used for HPLC are filtered through micron-size filters and degassed.
Removal of particulates prevents clogging of the inlet frit to the column and
removal of dissolved air minimizes the formation of bubbles that can interfere
with a flow-through spectrophotometer cell in the detector, or they can create
voids in the column that degrade column efficiency.
6. Samples separated by HPLC do not need to be easily vaporized.