CHEM 380
Practice EXAM III
Name
1. Describe at least three differences between HPLC and capillary electrophoresis.
2. Examine the chromatogram
below: A) identify the
retention time of each peak and
B) describe the resolution
difference between the peaks.
A) peak 1: 2.4 min, peak 2: 4.9 min,
peak 3: 5.8 min
B) The first peak is completely
resolved from the other peaks. The 2nd
and 3rd peaks are not resolved from
each other.
3. How does a Thermal Conductivity Detector (TCD) work? Draw a picture.
.
4. Draw and list the basic components of a capillary electrophoresis instrument.
5. How does Size Exclusion chromatography work (how does it move and separate analytes
through the column)?
6. What major problem does HPLC and CE suffer when each method uses an absorbance or
fluorescence detection method. How is this problem sometimes fixed?
HPLC and CE columns are small in diameter compared to a typical 1-cm cuvette. The directly
affects the b term in Beer’s Law (Abs=εbC), so the sensitivity in these methods suffers. One solution
to this problem is to use a Z-cell, which bends tube so that the pathlength is increased. This only
works if the resolution of the peaks is large, b/c if not then two analytles could be in the z-cell at the
same time and would not be resolved.
The other solution in fluorescence detection is to use a laser and vastly increase the incident power,
which increases sensitivity in the typical fluorescence eqn: F = K’P0C
7. Describe the typical mobile and stationary phase for each of the following chromatography
methods.
a. CE: capillary electrophoresis
b. GC: gas chromatography (open tubular column)
c. HPLC: high performance liquid chromatography
d. IC: ion chromatography
a) In CE, there is usually no stationary phase. The mobile phase is usually a buffered aqueous
solution.
b) In GC, the stationary phase is usually a thin coating on the inside wall of the tubing, which could
be made from a myriad of compounds for all kinds of different separations. The mobile phase is
an inert gas, such as He or N2.
c) In HPLC, the stationary phase is coated onto micro-beads made from polystyrene or other
polymer and packed into a steel tube. The mobile phase is a solvent or a mixture of two to four
solvents to achieve the right polarity in contrast to the stationary phase.
d) In IC, the stationary phase is a cationic or anionic resin. The mobile phase is an aqueous solution
of electrolytes, the pH and conductivity of which can be controlled during the experiment.
8. What is the van Deemter eqn, what is its purpose, and what does each term represent and how it
affect resolution?
H = A + B/u + C×u
Its purpose is to semi-quantitatively model the affect on the size of theoretical plates (H) that various
chromatographic parameters have. The A-term represents multiple paths and increases when the
analyte can move through different paths during elution and therefore experience a longer or shorter
elution without interacting with the stationary phase. This increases H and degrades resolution. The
B-term represents longitudinal diffusion. If temperature increases, then molecules with diffuse faster
into the mobile phase and peaks will get wider. The same thing happens with time. Diffusion can be
reduced by increasing the flow-rate, so the elution time is decreased. This will shrink H and improve
resolution. The C-term represents the mass transport term. It is proportional to time it takes the
analyte to reach equilibrium with the stationary phase. If the analyte is large or moves through a
viscous mobile phase, then interaction with the sp is limited/slow and the C term is large. Lowering
the flow rate will help this, but it also has a negative impact on the B term.