Ion-pair HPLC

1. When analyzing a drug X (strongly basic) and preservatives B (neutral), and C (strongly acidic), what would be the advantage of using IP-HPLC over regular RP-HPLC?

X retention would strongly increase, and the retention of C and B would slightly decrease [assuming counter ion is anionic – instructor’s comment].

The advantage of using IPC is that X, B, and C would be retained within a reasonable time, resulting in decrease in retention range.

2. When using ion-pair–HPLC to analyze a basic sample and the pH is decreased. What would be the effect in the retention of the sample and why?

Since pH was decreased, the retention time would increase because now the sample is more ionized making it possible for the sample to ion pair with the ion-pair reagent retained by the stationary phase.

3. What are 3 advantages of ion-pair-HPLC?

· IPC separation involves two additional variables that can be used for control of selectivity (IPC-reagent type and concentration). 

· Simple preparation of buffers. [instructor’s comment – not certain this is an advantage as it is more complex than most RP-HPLC]

· Wide choice of carbon chain lengths for improved retention and separation.

· Simultaneous separation of both ionized and non-ionized molecules.

· Improved peak shapes [instructor’s comment – vs. ionized molecules particularly for basic compounds]

4. What changes can be done during Reversed Phase Chromatography to increase retention of a compound that requires a mobile phase with pH>8 to improve retention (without trying the addition of IPC-reagent)?

The Use of a column that is stable at extreme pH.