High Performance Liquid Chromatography (HPLC)
Instructor: Dr. Hüseyin BOZKURT
High Performance Liquid Chromatography (HPLC) is one mode of chromatography, the most widely used analytical technique. Chromatographic processes can be defined as separation techniques involving mass-transfer between stationary and mobile phases. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC). HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components are first dissolved in a solvent, and then forced to flow through a chromatographic column under high pressure. In the column, the mixture is resolved into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute componentsand the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and has the ability to easily separate a wide variety of chemical mixtures.
In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more. High performance liquid chromatography (HPLC) makes use of a high pressure pump to deliver a mobile phase solvent at a uniform rate at pressures that are typically from 500 to 5000 psi. The most obvious advantage of HPLC over gravity liquid chromatography is that samples can be separated much more quickly. In addition, samples that are not volatile or that would thermally decompose in gas chromatography can be rapidly and routinely separated in HPLC. Consequently, this powerful analytical method is a great complementary instrument to gas chromatography.
High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds, and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.
HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also
provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase.
With HPLC, a pump (rather than gravity) provides the higher pressure required to propel th e mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography.
Schematic Diagram of a High Performance Liquid Chromatograph. (1) Solvent reservoirs,...