Homework Questions- Zirconia based stationary phases for HPLC
1. A chemist is running a column to separate proteins using a PBD-ZrO2 column. The mobile phase is composed of aqueous acetate buffer with THF as the organic modifier. Extremely broad peaks result. What is the most likely cause of the peak broadening and how can you change the mobile or stationary phase to help narrow the peaks? Hint: proteins contain carboxyl groups
a.
The
peak broadening is a result of the carboxyl groups of the proteins irreversibly binding strongly [irreversibly
would result in peak area decrease as it would not reach the detector – tailing
or broadending is usually due to more than one type of bonding – e.g. strong
and weak bonding – instructor edit] to the lewis acid sites on the zirconia
surface. Add fluoride or phosphate to the eluent to block lewis acid sites.
(Phosphate modified)
2. Name some differences between zirconia and silica and give 2 advantages over silica
a. Advantages over silica
i. Thermal stability
ii. Chemical stability
iii. Reversible modifications possible
iv. Amphoteric exchanger
v. Narrower particle size distribution
3. What is one way to increase elution efficiency on a zirconia based column if peaks are very well resolved?
a. Temperatures up to 200C may be used to increase elution efficiency. [Instructor’s Note: increasing T improves column efficiency (N) regardless of whether peaks are well or poorly resolved by increasing diffusion and decreasing the van Deemter C term. It also tends to decrease k values, so with poorly resolved peaks, the decrease in k could result in no change or a decrease in resolution despite an increase in N. The net effect of an increase of T is usually to shorten the time needed for a run.]
4. A chemist is trying to separate enantiomers. Which non-bonded zirconia modification could they use to achieve separation?