High-performance liquid chromatography
Summary
HPLC separates a mixture by pumping a liquid mobile phase at high pressure through a column of fine stationary-phase particles; compounds attracted more to the stationary phase elute later. Testable point: in normal phase the least polar compound elutes first, in reverse phase the most polar does.
Detail
Meaning: HPLC is column chromatography in which a liquid mobile phase is pumped at high pressure through a column packed with very fine stationary-phase particles. Compounds partition between the two phases. Those attracted more strongly to the stationary phase move more slowly and have longer retention times. A detector, often UV absorbance, records a peak as each compound exits the column.
On the MCAT: Expect a chromatogram (signal vs. time) and a question about elution order, or about how changing the column or solvent changes that order. In normal-phase HPLC the stationary phase is polar (silica) and the solvent is nonpolar, so the least polar compound elutes first. In reverse-phase HPLC the stationary phase is nonpolar (C18 hydrocarbon chains) and the solvent is polar, such as water with methanol, so the most polar compound elutes first. Peak area is related to the amount of each compound.
Common confusion: Students memorize "polar elutes first" or "nonpolar elutes first" without checking the column type. First identify which phase is polar. The compound most similar to the stationary phase is retained longest. Also note that HPLC uses a liquid mobile phase, unlike gas chromatography, where a gas carries the sample and separation depends largely on volatility and polarity.
Sources
- Organic Chemistry (Clayden, Greeves, Warren)
- Quantitative Chemical Analysis (Harris)
- Organic Chemistry (Wade)
Written for the MCAT and checked before it was published. For study reference only. Spotted an error? Let us know.