The hardware of DHPLC consists of components similar to those in conventional HPLC. Together with a gradient system, a pressure pump delivers buffers or solvents from buffer reservoirs in appropriate proportions to a column for the separation of DNA molecules. The buffer is the mobile phase while the column is the stationary phase.
DNA samples are placed in an autosampler plate and injected into the column through an injection unit. The column is housed in a temperature-controlled oven. Under appropriate conditions, DNA molecules are separated in the column into individual components and then eluted from the column. The eluted components are monitored by a detector and the data collected in a computer system. A UV detector is the most common option for measuring DNA molecules at a wavelength of 260 nm although a fluorescence detector can also be installed.
The results are displayed as chromatograms or elution profiles showing the amounts and elution times for various components separated by the column. An optional fragment collector can be connected to the detector to collect the separated components into vials for further analysis and processing. The heart of the system lies in the column—the stationary phase. The most widely used column is DNASep (Transgenomic).
DNA separating columns (e.g., Eclipse and Helix columns) from other manufacturers use a different type of stationary phase, and are less popular when compared with DNASep.