High performance liquid chromatography (HPLC) has become a powerful tool in analytical and preparative neurochemistry. Modern HPLC methods can be used for several purposes, including separation, purification and quantitative assessment of neurochemical compounds. It is important to understand the theoretical background to HPLC if it is to be used effectively. In brief, HPLC consists of two main components:
• A separation column or stationary phase. Compounds that contain functional groups capable of strong hydrogen bonding adhere more tightly than less polar compounds to the stationary phase. Thus, less polar compounds elute from the column faster than compounds that are highly polar.
• An aqueous mobile phase. The mobile phase in HPLC refers to the solvent which is continuously applied to the column, or stationary phase. The mobile phase is a buffered water/acetone solvent and exhibits polar properties.
The mobile phase is pumped through the column under high pressure (between 30 and 200 bar; or 3-20 MPa). When a sample is introduced into the system via an injector, it is dissolved in the mobile phase. The molecules in the sample separate according to their relative affinity to the non-polar matrix and the polar mobile phase. Molecules which are neutral or of low charge (more apolar) exhibit a greater affinity for the stationary phase and thus become trapped by the column, with the result that their elution is delayed. Molecules which are charged (and therefore more polar) possess a greater affinity to the mobile phase and elute faster. Increasing the length of the column or decreasing the particle size of the packing material improves separation, but also results in an increase in the pressure required to pump the mobile phase. When the column length is increased, the dilution of samples is also increased.
Preparative HPLC refers to process of isolation and quantitative purification of compounds. Important requirements for a successful preparation are the degree of solute purity and the "throughput', i.e. the amount of compound produced per time. In contrast in analytical HPLC, the focus is mainly on obtaining information about the composition of the sample. Recording the efflux of compounds from the HPLC column is a crucial part of any HPLC approach. In order to monitor the efflux, a UV detector must be selected and adjusted to optimal detection settings. Setting can be checked by standard separation assays and adjusted so that, in the standard samples, there is a sharp (detection) peak in the range expected from the substance being studied.
Quantification of compounds by HPLC is the process of determining unknown concentrations of a substrate. It involves the injection of a series of known concentrations of a standard compound into the HPLC column. The chromatogram of the known concentrations provides a series of peaks which correlate with the concentration of the injected compound. Normograms can be used to estimate unknown concentrations.
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