Problem Set

1. There are two forms of HPLC, normal phase HPLC and reversed phase HPLC. In normal
phase HPLC, the stationary phase is silica and the mobile phase is a non-polar solvent
like hexane. What are the stationary and mobile phases in reversed phase HPLC?

2. In normal phase HPLC, how will the retention time of a component of a mixture vary
with the polarity of its molecules? Explain your answer.

3. In reversed phase HPLC, how will the retention time of a component of a mixture vary
with the polarity of its molecules? Explain your answer.

4. One way of detecting components of a mixture coming out of the column is by using UV
light. Explain briefly how this works.

5. The retention time of a particular component of a mixture is measured from the time of
injection to its maximum peak height in the detector. What factors govern the length of
the retention time?

6. Explain briefly how a mass spectrometer can be used to identify the compounds
producing the peaks in high performance liquid chromatography.

7. The diagram shows a simplified output from the detector with peaks for two
compounds X and Y. The areas under the peaks are shown in green. What, if anything,
can you say about the relative concentrations of X and Y in the mixture? Explain your
answer.

8. Supercritical Fluid Chromatography (SFC) is more environmentally friendly than HPLC.

Why?
9. Refer to the three compounds listed above. Rank these compounds in order of
increasing retention time in a reverse phase liquid chromatography system.
Phenol, Naphthalene, 1-naphthol

10. This question refers to the two chromatograms shown below.

These chromatograms were recorded using reverse phase HPLC.

a. Give an example of a mobile phase that might be used for this type of separation
(i.e. reverse phase).
b. Give an example of a detector that might be used for these compounds.
c. Give an example of a stationary phase that might be used for these separations.
d. Chromatogram B was recorded on the same column and with the same sample as
chromatogram A, but the separation is very different. This is because the mobile
phase was
changed between the two separations. Explain how the mobile phase was changed
to affect the separation so dramatically. Be specific.
 Example answer for Q10.
a. Water/methanol, water/acetonitrile, etc.
b. UV-Vis absorption – they are all aromatic organic compounds
c. C18 (ODS), C8, C4, Phenyl are all acceptable
d. Chromatogram B was recorded using a mobile phase with a lower eluent strength
than
chromatogram B. For example, Chromatogram A may have been recorded in 90%
Methanol and 10% water, whereas Chromatogram B may have been recorded in
50% Methanol and 50% water. You must mention that the % organic modifier in
Chromatogram A is greater than that in Chromatogram B.

11. List the following HPLC detectors in order from lowest to highest detection limits:
UV-Visible detector
Evaporative light scattering detector
Fluorescence detector

Example answer for Q11

Lowest: Fluorescence detector
Highest: Evaporative light scattering detector

12. Choose the one correct answer. You are analyzing a sample by HPLC that contains
both neutral analytes and ionized analytes that cannot be converted to the neutral form
using
buffers. What HPLC technique should you use for the analysis?
Example answer for Q12
Reverse phase HPLC with ion-pairing reagents

13. Your sample to be analyzed by reverse phase HPLC contains an analyte that is an
acid (HA) with a pKa of 4.46. To what pH should you buffer your aqueous eluent in order to have
an HA to A- ratio of 100: 1 in the aqueous phase?

[base]
pH= pKa+log
[acid ]
pH=2.46