Question

1. In Normal-phase HPLC (hexane is used as the mobile phase), which one elute first and which one elute last? Sample mixture consist of Phenol, Toluene, and Ethylbenzene. Explain the elution order of the components in HPLC in terms of functionalities of the compound mixture.

2. If different mobile phase has used instead of hexane, how it will affect elution order and/or elution time?

Answer 1

1. The compounds will be separated on the basis of polarity. Here the mobile phase used is hexane. It is a non-polar solvent. Hence the least polar will be separated out first. The compounds will be separated from the least polar to the most polar.Most polar will stay longer and will separate last. The most polar compound here is phenol. Hence it is the last one to get eluted. Toluene is more polar than ethyl benzene. Hence the first one to get eluted is the least polar ethylbenzene.

The order of elution is thus ethylbenzene, toluene and phenol respectively. Here ethylbenzene is the first to get eluted and phenol is the last one.

2. If a different solvent i.e., a solvent with more polarity is used, the elution time decreases. If the mobile phase is highly polar all the components will be eluted at once and hence we will obtain a mixture. Depending on the compounds to be eluted, the solvent should be chosen based on polarity. Otherwise a proper elution will not occur. The purity of the samples will thus be affected.