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For Liquid Chromatography
High-performance liquid chromatography is an efficient type of column chromatography widely used in pharmaceuticals. It is very useful for determination of assay and related substances in drug substance. In general, HPLC is used to separate the components of a mixed drug substance.
In HPLC chromatography, the column plays an important role in the separation of various compounds because it contains the stationary phase. The stationary phase is either polar or non-polar particles depending on the type of column. Polar and non-polar columns are used depending on the nature of the sample to be analyzed.
A mechanical pump is used to pump the mobile phase into the system and the injector injects the sample into the mobile phase which enters the column at a constant flow rate. The mobile phase acts as a carrier throughout the process.
When a mixture of compounds enters a column, it separates the compounds based on their polarity. If the stationary phase is non-polar then it attracts the non-polar compound and a polar compound is released first then a non-polar compound and if the stationary phase is polar then the non-polar compound is released first.
Detectors are used to determine individual compounds by ultraviolet absorption. Light absorption depends on the concentration of the compound in the mobile phase. These reaction signals are recorded in peak form by computer software and compound purity is calculated by peak area of different compounds. Computer systems and software should be verified.
