HPLC-Detectors (DAD)

HPLC is a physical method to separate, identify and quantify substances by distribution between the mobile, moving and the stationary, steady phase.

Functionality of HPLC

HPLC, high performance liquid chromatography, is a separation process where the liquid sample is carried by the liquid phase through the separation column over the stationary phase, on account of high pressure. Length and diameter of the separation column vary according to application. In some cases, a precolumn is placed before the HPLC column to remove impurities before separation. In contrast to gas chromatography, no gaseous or non-decomposed evaporateable substances are necessary for HPLC. In the case of strong interaction of the analyte with the stationary phase, the analyte remains in the column for quite a long time. If there is weak interaction, the opposite case is true: The analyte exits the column quite quickly. Therefore the substances appear at the end of the separation column after different times, the so-called characteristic retention times. HPLC can be differentiated by two principles: normal phase and reversal phase chromatography. In the case of NP-HPLC, a polar stationary phase is used, for example, silicon gel. In this process, the polarity determines the elution force of the mobile phase. The solvents are getting arranged by rising polarity, resulting in the so-called eluotropic series. This means that substances with higher polarity elute faster. Polar molecules better interact with the polar stationary phase and remain therefore longer in the column. RP-HPLC applies a non-polar stationary phase. The elution force decreases with rising polarity.

Classification of HPLC

In HPLC, several separation mechanisms can be distinguished, depending on the kind of interaction between stationary phase, mobile phase and the sample: adsorption, partition, ion exchange, size exclusion and bioaffinity chromatography. However, adsorption and partition chromatography are the most common modes of HPLC.

Architecture of an HPLC system

An HPLC system consists of an eluent vessel, a degasser, a pump, an injection valve, a precolumn, a column, a detector and an evaluation unit.

Literature

• http://de.wikipedia.org/w/index.php?title=Hochleistungsfl%C3%BCssigkeitschromatographie&oldid=86523806 (called: 30.03.11).