In reverse-phase HPLC with a C18 column and an aqueous/acetonitrile mobile phase, two compounds are injected: one is polar and hydrophilic, the other is nonpolar and hydrophobic. Which will elute first, and why?
AThe nonpolar compound, because it is repelled by the polar aqueous mobile phase and moves faster
BThe polar compound, because it has low affinity for the nonpolar stationary phase and is carried through quickly by the aqueous mobile phase
CBoth elute at the same time, since retention time depends only on molecular weight
DThe polar compound, because polar interactions with the C18 phase are stronger than hydrophobic ones
In reverse-phase HPLC, the stationary phase (C18) is nonpolar and the mobile phase is predominantly aqueous. Polar, hydrophilic compounds have little affinity for the nonpolar stationary phase and are swept through quickly, eluting early. Nonpolar compounds interact strongly with C18 via hydrophobic interactions and are retained longer, eluting later. Retention time reflects partitioning between stationary and mobile phases, not molecular weight.
Question 2 True / False
If two compounds have the same retention time in an HPLC run, you can confidently conclude they are the same compound.
TTrue
FFalse
Answer: False
Retention time is necessary but not sufficient for identification. Different compounds can co-elute under a given set of conditions. Definitive identification requires additional spectral data — a UV/Vis diode array detector showing the same absorption spectrum, or mass spectrometric detection confirming the molecular ion and fragmentation pattern. This is one of the most common over-interpretations in HPLC practice.
Question 3 Short Answer
What is gradient elution in HPLC, and what problem does it solve when analyzing a complex mixture?
Think about your answer, then reveal below.
Model answer: Gradient elution progressively increases the proportion of organic solvent (e.g., acetonitrile) in the mobile phase over the course of the run. Early-eluting polar compounds are separated under weak conditions; as the gradient increases organic content, more hydrophobic compounds are pulled off the column. Without gradient elution, a fixed mobile phase composition either fails to retain early peaks adequately or requires very long run times to elute late-eluting compounds.
In complex samples, analytes span a wide range of hydrophobicities. Isocratic (fixed composition) conditions cannot optimize resolution and run time simultaneously for all components. Gradient elution is analogous to adjusting the 'strength' of the mobile phase mid-run to sweep out progressively more retained analytes efficiently.