BSK3123 SEPARATION TECHNIQUE

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

TUTORIAL

1. Describe three general methods for improving resolution in partition chromatography.

2. What is the order of elution of the following compounds from a normal-phase packed HPLC column?
(a) Ethyl acetate, acetic acid, dimethylamine
(b) Propylene, hexane, benzene, dichlorobenzene

3. What is the order in which the following compounds would be eluted from an HPLC column containing
reverse phase packing?
(a) Benzene, diethyl ether, n-hexane
(b) Acetone, dichloroethane, acetamide

4. In preparing a hexane-acetone gradient for an alumina high performance liquid (HPLC) column, is it
desirable to increase or decrease the proportion of hexane as the column is eluted?

5. Define
(a) Isocratic elution
(b) Gradient elution
(c) Reverse-phase packing
(d) Normal phase packing
(e) Ion-pair chromatography
(f) Ion chromatography
(g) Sparging

6. What is guard column and what does it do?

7. In what way are normal-phase partition chromatography and adsorption chromatography similar?

8. What type of species can be separated by HPLC but not gas chromatography (GC)?

9. Mass spectrometry (MS) is an extremely versatile detection system for GC. However, interfacing it with an
HPLC is a much more difficult task. Describe the major reason why it is more difficult to combine HPLC with
MS that it is to combine GC with MS.

10. Although temperature does not have nearly the effect on HPLC separation that it has on GC separations, it
nonetheless can play important role. Discuss how and why temperature might or might not influence the
following separation
(a) A reverse-phase chromatographic separation of a steroid mixture
(b) An adsorption chromatographic separation of a mixture of closely related isomers
11. An HLPC method was developed for the separation and determination of ibuprofen in rat plasma samples as
part of a study of time course of the drug in laboratory animals. Several standards were chromatographed
and the following results obtained:
Ibuprofen concentration
Relative peak area
(g/mL)
0.5 5.0
1.0 10.1
2.0 17.2
3.0 19.8
6.0 39.7
8.0 57.3
10.0 66.9
15.0 95.3

Next, a 10 mg/kg sample of ibuprofen was administrated orally to a laboratory rat. Blood samples were
drawn at various times after administration of the drug and subjected to HPLC analysis. The following
results were obtained:
Time (hr) Relative peak area
0 0
0.5 91.3
1.0 80.2
1.5 52.1
2.0 38.5
3.0 24.2
4.0 21.2
6.0 18.5
8.0 15.2

Find the concentration of ibuprofen in the blood plasma for each of the times given above and plot the
concentration versus time. On the percentage basis, during what half-hour period (first, second, third etc.) is
most of the ibuprofen lost?

( p/s: Don’t have graph paper? http://incompetech.com/graphpaper/plain/ )