High performance liquid chromatography (HPLC) has become a standard and routine method for separation and concentration of analyses. An HPLC system consists of a solvent reservoir, high pressure pumps, sample injection port, separation column, detector, and recorder units. HPLC is the result of attempts to increase the efficiency of liquid chromatography to the same level as that of gas chromatography.
Image Source: cpiinternational.com/media/wysiwyg/shutterstock_95733688.jpg
This is achieved by using particles of 2-20 pm size to overcome diffusion-related problems in the liquid and solid phase. The resulting high inlet pressure in the column is overcome by resorting to high pressure pumping of solutions. A pump forces one or more mobile phases through the tightly packed high efficiency column. The sample is introduced through an injection system into the entrance to the column.
Sample introduction, can be improved by flow-injection method, by injecting the sample into a flowing stream which carries the analyte through a chemical modulator into a detector. There is no universal detector for HPLC. Detectors based on refractive index (RI), optical absorbance, fluorescence, thermal conductivity, and flame ionization are in use.
ADVERTISEMENTS:
Reverse-phase HPLC (RP-HPLC) is very well suited for the analysis of non-volatile polar compounds, such as polycyclic aromatic hydrocarbons (PAHs). Adsorbed PAHs on filters are recovered using Soxhlet extraction. The sample is then separated into fractions by column chromatography on silica gel using hexane-chloroform as the mobile phase, and individual components are detected by fluorescence. Advent of HPLC has resulted in tremendous progress in the separation of a wide variety of inorganic and organic compounds, with high sensitivity of detection.