Components of HPLC Analysis

HPLC has four basic components: a solvent delivery system to provide the driving force for the mobile phase; a means by which samples can be introduced into the solvent; the column; and some type of detector. A recorder is used to display the results and an integrator performs the calculations.

The column used for a specific separation is based on the type of the molecules to be analyzed. Various types of chromatographic modes can be used for the separation of the molecules. For example, ion exchange columns separate charged molecules such as amino acids, proteins, or nucleotides. Size exclusion columns separate organic polymers such as polyvinyls and silicones or biopolymers such as proteins, nucleic acids, or sugars. Adsorption columns separate molecules based on their interaction with the stationary phase. This mode is useful for the separation of vitamins, dyes, lipids, phenols, and antioxidants. Partition columns are used to separate molecules based on the way that the solvent becomes partitioned into stationary and mobile layers, and is useful in analyzing steroids, aromatics, vitamins, and antibiotics. The molecules eluting from any one of these different types of column are then analyzed by various types of detectors, measuring absorbance, fluorescence, or electrochemical or radiochemical properties. Other types of detectors include mass spec-troscopy and refractive index.

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