HPLC (High-Performance Liquid Chromatography) is a chromatographic technique used to separate mixtures or compounds in the field of analytical chemistry, biochemistry and in the manufacturing sector. HPLC thus plays a critical role in the pharmaceutical field since its used to test the products and ingredients used to make them. Simply said, it’s a method of separating, identifying and quantifying each component in a mixture or compound. It involves the use of pumps to pass pressurized liquid solvent containing a sample of the mixture through a column containing solid absorbent material. An absorbent is a substance that attracts another substance or particle to its surface.
The principle of HPLC is not a complicated rocket science to understand. The sample mixture to be separated and analyzed is introduced, in small volumes, in the mobile phase percolating through the column. The mobile phase is the pressurized liquid which is typically a mixture of solvents like water, methanol and acetonitrile. Mechanical pumps are used to pump the mobile phase into the system and injector introduces the sample to be analyzed into the mobile phase which enters the column at a constant mass flow rate. However, components of the mixture move through the column at different velocities which are functions of their interaction with the absorbent material. The column separates the components of the compound or mixtures on the basis of their polarity. If stationary phase(absorbent) is nonpolar, it attracts the non-polar components and the polar components elute first then a non-polar component. If the absorbent is polar, the non-polar component elutes first.
The velocity of each component depends on its chemical nature, nature of the column and composition of the mobile phase. The time after which a specific analyte emerges from the column is called retention time and is an identifying characteristic of a given analyte when measured under particular conditions. An analyte is a sample whose constituents are being examined.
Various types of columns are available filled with absorbents varying in sizes of particles and the nature of the surfaces. Use of small size particles needs high pressure during operation and it improves the chromatographic resolution (the degree of separation of consecutive analytes emerging from the column. Common mobile phases used are any miscible mixture between water and inorganic solvents like acetonitrile and methanol. Some HPLC uses water-free mobile phases. The aqueous component of a mobile phase may contain acids or salts to help in separation of components. Also, the composition of the mobile phase may be kept constant or may be varied during the chromatographic analysis. Detectors are used to identify the separated components by ultraviolet light absorption which depends on the concentration of the mixture in the mobile phase.
The principle of HPLC outlined above can be done in various methods namely, partition chromatography, normal-phase chromatography, displacement chromatography, reversed-phase chromatography (RPC), size-exclusion chromatography and ion-exchange chromatography. The parameters to consider for the mentioned types are the internal diameter of the column, the pore size of absorbent, pump pressure, particle sizes and autosamplers.
The principle of HPLC has a major effect in the pharmaceutical field and its advantages and efficiency cannot be parallel to any other method of determining the ingredients or components of compounds and mixtures used in the manufacture of products like drugs.