In practice, the analysis begins with a standard polymerase chain reaction (PCR) in order to amplify the fragment of interest.
Heteroduplexes are thermally less stable than their corresponding homoduplexes, and the single DNA strands will therefore be disconnected by chromatography when subjected to a sufficiently high temperature.
This mismatch will therefore decrease the interaction with the column and will result in a reduced retention time compared to the homoduplexes in the chromatographic separation process.
To observe the phenomenon of separation, the DHPLC method uses a column of a non-grafted porous stationary phase composed of polystyrene-divinylbenzene alkyl.
The positively charged ammonium ion of these molecules interacts with the DNA, and the alkyl chain with the hydrophobic surface of the solid phase.