High performance liquid chromatography (HPLC) is a separation technique that uses liquid mobile phase to separate the components of a mixture. The sample is first dissolved in a solvent, then forced to flow through a chromatographic column under high pressure. Within the column, the mixture is resolved into its component parts. Individual HPLC columns may use any one of a number of different phases or processes to induce this resolution including normally bonded phase, reversed phase, size exclusion, ion exclusion, and ion exchange.
Normally bonded phase HPLC columns are composed of a stationary bed, which is strongly polar in nature, and a mobile phase that is non-polar. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials. This technique works well with water-sensitive samples, analytes that are insoluble in water, certain geometric isomers that are difficult to separate by reversed-phase, and class separations. Organic solvents used in normal-phase separations are more Mass Spectroscopy (MS) friendly than some of the typical buffers used in HPLC. In reversed-phase HPLC, operating conditions sometimes must be dramatically modified to allow compatibility with MS detection.
Reversed phase HPLC columns are the inverse of normal phase chromatography. The stationary bed is non-polar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. The lower the polar differential, the longer the material is retained.
In size exclusion the HPLC column is filled with material having precisely controlled pore sizes, and the sample is screened or filtered by the stationary phase according to its solvated molecular size. Larger molecules are rapidly washed through the column, and smaller molecules penetrate the pores of the packing particles and elute later.
In ion exclusion the anions are retained longer on the stationary phase than on anion-exchange columns. Quantification is therefore simplified in the event large amounts of cationic contaminants are present in the sample.
In ion exchange, the stationary bed with the HPLC column has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.