Question

HPLC problem:

Would you increase or decrease the time it takes to change the mobile phase gradient from 40 % acetonitrile/60 % aqueous buffer to one that is 90% acetonitrile/10% aqueous buffer if the goal is to elute the more non-polar riboflavin in a shorter retention time using a C18 column?

Answer 1

Riboflavin is non-polar and C18 column is less polar as compared to silica. Hence, riboflavin is strongly retained by C18 and elutes later, thereby, giving a higher retention time.

The initial mobile phase composition is 40% acetonitrile-60% aqueous buffer. The mobile phase composition changes to 90% acetonitrile-10% aqueous buffer during the course of the separation. Acetonitrile is less polar than water and increasing the concentration of acetonitrile in the mobile phase decreases the polarity of the mobile phase. Consequently, non-polar or less polar compounds are attracted to the less polar solvent now and elute out.

Therefore, in order to reduce the retention time of Riboflavin, the mobile phase gradient must be changed quickly. Therefore, we must decrease the time to affect the mobile phase gradient change. However, it must be noted that rapidly changing the mobile phase gradient will cause all non-polar compounds to elute out of the column and contamination may occur. Hence, we must decrease the time taken only when we are sure that Riboflavin is the most non-polar compound retained on the column and there are no other compounds to co-elute with Riboflavin.