Question

1. Why was it important to filter and degas the mobile phase and the samples?

2. How is the “reversed-phase” HPLC used here different from “normal-phase” with regard to

   stationary and mobile phases, and order of elution?

3. Mobile Phase Composition

   (a) If the mobile phase composition was changed from 65:35:1 (v/v/v) to 75:25:1 (v/v/v) water:methanol:acetic acid, how would the time of elution (expressed as retention time) for caffeine be changed, and why would it be changed?

(b) What if it was changed from 65:35:1 (water:methanol:acetic acid) to 55:45:1? How would that change the retention time and why?

Answer 1

1) It is important to filter and degassing Mobil phase and the sample ,as if there is any particles in mobile phase it will make interference with the pressure of mobile phase or it will block cloumn as a result the functionality of column will reduce.

If we don't deggass mobile phase the bubble will trapped in the column and pressure will drop as a result the anlysis results will not be proper.

2) In reverse phase HPLC the stationary phase is organic or non polar and the mobile phase is polor or aqueous. As a result organic ( non polar) molecules will retain for more time that the polar molecules.

Polor molecules will elute first then organic molecules.

a) If the if the mobile phase composition is changed from 65:35:1 to 75:25:1 water ,methaol ,acetic acid

The ploar composition will increase ,and as caffeine is soluble in polar solvent hence its retention time will be increased

As reverse phase HPLC the stationary phase is non plolr .it will have more affinity toword polar molecules hence it will elute relatively in less time.

b) If the composition is changed from 65:35:1 to 55:45:1

The non polar composition will increase ,this will decrease the retention time if caffeine.

As composition of the phase will be more non polar molecules will spend less time in the column or so molecules can easily elute.